Method for production of reprogrammed cell using chromosomally unintegrated virus vector

ABSTRACT

An objective of the present invention is to provide vectors for conveniently and efficiently producing ES-like cells in which foreign genes are not integrated into the chromosome. The present inventors discovered methods for producing ES-like cells from somatic cells using chromosomally non-integrating viral vectors. Since no foreign gene is integrated into the chromosome of the produced ES-like cells, they are advantageous in tests and research, and immunological rejection and ethical problems can be avoided in disease treatments.

TECHNICAL FIELD

The present invention relates to methods for producing reprogrammedcells, cells produced by these methods, compositions used in thesemethods, and such. In particular, the present invention relates tomethods for producing pluripotent stem cells from differentiated somaticcells, and pluripotent stem cells prepared by such methods.

BACKGROUND ART

Embryonic stem cells are stem cells established from the inner cell massof mammalian blastocysts, and they can be proliferated infinitely whilemaintaining the ability to differentiate into all types of cells(differentiation pluripotency). This property anticipates stem celltherapy or myocardial infarction, Parkinson's disease patients, or such,which is achieved by transplanting myocardial cells or nerve cellsinduced and prepared in large quantities from ES cells. Furthermore,uses in basic pathological and pharmacological studies and as adevelopment tool in drug discovery are also anticipated. However, theseES cells have the ethical problem of utilizing and sacrificing humanfertilized eggs. There is also the problem of immune rejection where thehistocompatibility antigens of limited donor fertilized eggs do notmatch with the patient. On the other hand, tissue stem cells such asneural stem cells, hematopoietic stem cells, and mesenchymal stem cellsare present in every tissue of the living body. Since tissue stem cellsdo not use fertilized eggs, there are few or no ethical problems, andsince cells of the patients themselves can be used, immune rejectionreactions can also be avoided. However, properties of tissue stem cellsare not necessarily understood, and therefore they are difficult toisolate, and their numbers are also very few. Their proliferativeability and differentiation ability are also much more limited comparedto ES cells. If somatic cells such as tissue stem cells anddifferentiated cells can be converted by some means into cells similarto ES cells having a high proliferative ability and differentiationpluripotency (referred to as ES-like cells), such ES-like cells will beideal stem cells in clinical applications and such.

Specifically, cells of mammals, particularly somatic cells of patients(tissues of the skin, stomach or lung, blood cells, and such) arecollected, and these cells are cultured and then stimulated with nuclearreprogramming factors (factors that induce nuclear reprogramming) toproduce ES-like cells (they may also be called “artificial pluripotentstem cells”, “induced pluripotent stem cells (iPS cells)”, or “embryonicstem cell-like cells”). These produced cells are expected to be appliedclinically as stem cells or used in basic research includingpharmacological or pathological research (Patent Document 1) just asthey are, or after storage in cell banks. Furthermore, experiments toconfirm pharmaceutical effects can also be carried out using artificialpluripotent stem cells established from patients.

Examples of nuclear reprogramming factors include the Oct gene, the Klfgene, the Myc gene, the Sox gene, the Nanog gene, the Lin28 gene, theTERT gene, and the SV40 Large T gene (Patent Document 2, Non-PatentDocuments 1 to 7).

For example, it is known that the above-mentioned ES-like cells can beproduced from the above-mentioned somatic cells using the following fourrecombinant virus vectors (Non-Patent Document 1 to 7). When theproduced ES-like cells described above are used clinically, they may beable to avoid problems of immune rejection and ethical problems.

(1) gamma retroviral vector or lentiviral vector (hereinafter, thesevectors will be collectively referred to as “retroviral vectors”)containing the Oct3/4 gene

(2) retroviral vector containing the Klf4 gene

(3) retroviral vector containing the c-Myc gene

(4) retroviral vector containing the Sox2 gene

The above-mentioned patent documents and non-patent documents are asfollows:

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: International Publication WO 2005/080598-   Patent Document 2: International Publication WO 2007/069666

Non-Patent Documents

-   Non-Patent Document 1: Cell. 2007 Nov. 30; 131(5):861-872-   Non-Patent Document 2: Science. 2007 Dec. 21; 318(5858):1917-1920-   Non-Patent Document 3: Nat Biotechnol. 2008 January; 26(1):101-106-   Non-Patent Document 4: Science. 2007 Dec. 21; 318(5858):1920-1923-   Non-Patent Document 5: Nature. 2008 Jan. 10; 451(7175):141-146-   Non-Patent Document 6: PNAS. 2008 Feb. 26; 105(8):2883-2888-   Non-Patent Document 7: Cell. 2008 Apr. 18; 133(2):250-264

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

However, it should be noted that the ES-like cells produced using theabove-mentioned retroviral vectors have their chromosomes structurallymodified by integration of the vectors into the host chromosomes. Theymay have unanticipated abnormalities in chromosomal functions, and inparticular the cells may become cancerous. The reason is becauseretroviral vectors are used. When retroviral vectors are used, there isthe risk that random integration of the vectors into the chromosomes ofthe transduced cells might cause inactivation of tumor suppressor genesin the chromosomes or activation of genes involved in cancer formationnear the insertion site (Jikken Igaku (Experimental Medicine) Vol. 26,No. 5 (supplement): pp. 35-40, 2008). Furthermore, when they areintegrated into other genes or genes that modify the expression of thosegenes, the cells may change into cells with unexpected properties. Inaddition, since the so-called noncoding regions of the chromosome arerecently considered to have certain chromosomal functions as well,unfavorable consequences brought about due to integration of theretroviral vectors into the noncoding regions must also be considered.Additionally, in the case vectors are inserted into genes involved inthe differentiation of the pluripotent stem cells, there is apossibility that treatments or studies using the cells obtained bydifferentiation of the stem cells cannot be carried out, sincedifferentiation does not occur and cells cannot be obtained.Accordingly, with cell reprogramming by conventional methods, problemsof safety remain in treatments that use the obtained ES-like cells. Inaddition, in drug efficacy and pathological analyses using ES-like cellsestablished from patients, effects caused by inactivation or activationof genes originally functioning in cells as a result of insertion offoreign genes into the chromosome must be considered, and such analyseswill become extremely difficult operations. Furthermore, when retroviralvectors are used, the established ES-like cells will have the retroviralvectors inserted into different sites in the chromosome depending on thelot even when production is carried out by the same researcher or bydifferent producers according to the same protocol; therefore, there isalso the problem that homogeneity of artificial pluripotent stem cellscannot be guaranteed.

The present invention was accomplished with the objective offundamentally solving such situations, and provides methods for easilyand efficiently producing ES-like cells in which foreign genes are notintegrated into the chromosome, i.e. chromosomally non-integrating EScells. Furthermore, the present invention provides gene transfercompositions that are useful for inducing reprogramming by theabove-mentioned methods. The present invention also provides pluripotentstem cells obtained by the methods of the present invention.

Means for Solving the Problems

The present inventors discovered that pluripotent stem cells in whichforeign genes are not integrated into the chromosomes can be produced byusing vector types with no chromosomal integration.

That is, the present invention relates to methods for producingpluripotent stem cells using chromosomally non-integrating vectors,ES-like cells produced by the methods of the present invention, andsuch, and more specifically relates to the inventions described in eachof the claims. Inventions consisting of any combination of two or moreinventions described in claims that cite the same claim are alsoinventions intended herein. Accordingly, the present invention relatesto the following:

[1] A method for introducing a gene into a cell to reprogram the cell,wherein the gene is introduced into the cell using a chromosomallynon-integrating viral vector.

[2] The method of [1], wherein the reprogramming is induction of apluripotent stem cell.

[3] The method of [1] or [2], wherein the chromosomally non-integratingviral vector is an RNA viral vector.

[4] The method of [3], wherein the RNA viral vector is a minus-strandRNA viral vector.

[5] The method of [4], wherein the minus-strand RNA viral vector is aparamyxovirus vector.

[6] The method of [5], wherein the paramyxovirus vector is a Sendaivirus vector.

[7] The method of any one of [1] to [6], wherein the gene is selectedfrom the group consisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

[8] A composition for use in gene introduction for reprogramming a cell,which comprises a chromosomally non-integrating viral vector.

[9] The composition of [8], wherein the reprogramming is induction of apluripotent stem cell.

[10] The composition of [8] or [9], wherein the chromosomallynon-integrating viral vector is an RNA viral vector.

[11] The composition of [10], wherein the RNA viral vector is aminus-strand RNA viral vector.

[12] The composition of [11], wherein the minus-strand RNA viral vectoris a paramyxovirus vector.

[13] The composition of [12], wherein the paramyxovirus vector is aSendai virus vector.

[14] The composition of any one of [8] to [13], wherein the gene isselected from the group consisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

Moreover, the present invention relates to the following:

[1] A method for producing a reprogrammed cell, which comprises the stepof contacting a differentiated cell with at least one chromosomallynon-integrating viral vector.

[2] The method of [1], wherein the reprogrammed cell is an artificialpluripotent stem cell.

[3] The method of [1] or [2], wherein the vector is at least onechromosomally non-integrating viral vector that carries at least onegene encoding a nuclear reprogramming factor.

[4] The method of [3], wherein the gene is selected from the groupconsisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

[5] The method of any one of [1] to [4], wherein the vectors are used incombination so that at least the three genes, Oct, Klf, and Sox genes,or at least the four genes, Oct, Sox, Nanog, and Lin28 genes, areexpressed in a cell endogenously or exogenously.[6] The method of [5], wherein the vectors are used in combination sothat at least the four genes, Oct, Klf, Sox, and Myc genes are expressedin a cell endogenously or exogenously.[7] The method of any one of [1] to [6], wherein the chromosomallynon-integrating viral vector is an RNA viral vector.[8] The method of [7], wherein the RNA viral vector is a minus-strandRNA viral vector.[9] The method of [8], wherein the minus-strand RNA viral vector is aparamyxovirus vector.[10] The method of [9], wherein the paramyxovirus vector is a Sendaivirus vector.[11] A method of producing a differentiated cell, which furthercomprises the step of differentiating a cell produced by the method ofany one of [1] to [10].[12] A cell produced by the method of any one of [1] to [11].[13] The cell of [12], wherein the vector is not integrated into thechromosome in the step of reprogramming.[14] A composition for use in reprogramming of a cell, which comprises achromosomally non-integrating viral vector as an expression vector.[15] The composition of [14], wherein the reprogramming is induction ofa pluripotent stem cell.[16] The composition of [14] or [15], wherein the chromosomallynon-integrating viral vector is an RNA viral vector.[17] The composition of [16], wherein the RNA viral vector is aminus-strand RNA viral vector.[18] The composition of [17], wherein the minus-strand RNA viral vectoris a paramyxovirus vector.[19] The composition of [18], wherein the paramyxovirus vector is aSendai virus vector.[20] The composition of any one of [14] to [19], wherein the vectorcarries at least a reprogramming factor-encoding gene which is selectedfrom the group consisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

[21] Use of a chromosomally non-integrating viral vector in theproduction of an agent for reprogramming a differentiated cell.

[22] The use of [21], wherein the reprogramming is induction of apluripotent stem cell from a differentiated cell.

[23] The use of [21] or [22], wherein the chromosomally non-integratingviral vector is an RNA viral vector.

[24] The use of [23], wherein the RNA viral vector is a minus-strand RNAviral vector.

[25] The use of [24], wherein the minus-strand RNA viral vector is aparamyxovirus vector.

[26] The use of [25], wherein the paramyxovirus vector is a Sendai virusvector.

[27] The use of any one of [21] to [26], wherein the vector carries atleast a gene encoding a reprogramming factor which is selected from thegroup consisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

Further, the present invention relates to the following:

[1] A chromosomally non-integrating viral vector, which carries a geneselected from the group consisting of:

(1) the Oct gene;

(2) the Klf gene;

(3) the Myc gene;

(4) the Sox gene;

(5) the Nanog gene;

(6) the Lin28 gene;

(7) the SV40 Large T antigen gene; and

(8) the TERT gene.

[2] The vector of [1], wherein the chromosomally non-integrating viralvector is an RNA viral vector.

[3] The vector of [2], wherein the RNA viral vector is a minus-strandRNA viral vector.

[4] The vector of [3], wherein the minus-strand RNA viral vector is aparamyxovirus vector.

[5] The vector of [4], wherein the paramyxovirus vector is a Sendaivirus vector.

Any components of the inventions described herein and any combinationthereof are intended herein. In these inventions, inventions excludingany components described herein, or any combinations thereof are alsointended herein. Furthermore, certain specific embodiments describedherein regarding the present invention not only disclose theseembodiments, but also disclose inventions excluding these embodimentsfrom generic inventions disclosed herein which include theseembodiments.

Effects of the Invention

As described above, since cells produced by the methods of thisinvention do not have foreign genes incorporated into the chromosomes,they are not only advantageous in tests and studies utilizing thesecells, but can avoid immune rejection problems or ethical problems inthe treatment of diseases. Further, they can also help avoid the risk ofgenotoxicity-based transformation, unexpected side-effects due toalterations in chromosomal functions, and alterations of cellularproperties. Furthermore, pluripotent stem cells can be induced fromdesired cell types including adult skin cells with significantly higherefficiency (for example, approximately 10 times) using the methods ofthe present invention, compared to conventional methods usingretroviruses. Furthermore, with conventional methods using retroviruses,even if cells are produced by completely identical protocols, theES-like cells that are established will have the retroviral vectorsinserted into different sites in the chromosome; thus, the homogeneityof the artificial pluripotent stem cells cannot be guaranteed. Incontrast, with the methods of the present invention, since the vectorsare not inserted into the chromosomes, cells that are more geneticallyhomogeneous can be stably produced. Furthermore, retroviruses generallyhave strong tropism, and for example, with ecotropic retroviral vectorscurrently used in common reprogramming methods, presence of retrovirusreceptors, or their introduction from outside, becomes necessary priorto reprogramming, and establishment of artificial pluripotent stem cellsin animal species not expressing them has been difficult. In contrast,the methods of the present invention can be applied to a wide range ofanimal species (mammals in general). For example, this enablesapplication to biological species in strong demand as disease modelanimals, such as monkeys and pigs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs indicating the morphology of cells obtained bythe methods according to the present invention. The panels at the centerand on the right in the top row show the colonies 23 days after vectorintroduction. The panels at the bottom row show the passaged colonies.

FIG. 2 shows the results of staining of the cells obtained by themethods according to the present invention by alkaline phosphatase.

FIG. 3 shows the intracellular expression levels of specific genes ofthe cells obtained by the methods according to the present invention.The results of RT-PCR using mRNA prepared from the alkalinephosphatase-positive colony group (ALP(+)) (panel (a)) and mRNA preparedfrom a single colony (panel (b)) are shown. In these cells, expressionof Oct3/4, Sox2, Klf4, and c-Myc were confirmed, and in addition,expression of Nanog which is an ES cell marker was also observed (panels(a) and (b)). Furthermore, in cells passaged from a single clone,expression of hTERT which is a telomerase activation indicatorindicating infinite proliferation ability was observed (panel (b)). BJ:Cells not introduced with a vector. NCCIT: fetal carcinoma cells(positive control). The control is a negative control without templateDNA.

FIG. 4 shows the expression of ES marker in the cells obtained by themethods according to the present invention. BJ: Cells not introducedwith a vector. NCCIT: fetal carcinoma cells (positive control). Thecontrol is a negative control without template DNA.

FIG. 5 shows the results of detecting telomerase activity in cellsobtained by the methods according to the present invention.

FIG. 6 shows the pluripotency of the cells obtained by the methodsaccording to the present invention. Results of experiments on embryoidbody formation are shown.

FIG. 7 shows the pluripotency (in vitro) of the cells obtained by themethods according to the present invention. It shows in vitrodifferentiation of virus-free induced pluripotent stem (iPS) cellsinduced by the vectors of the present invention from human BJ cells intouseful cells derived from the mesoderm (myocardial and blood cells),ectoderm (TH-positive dopamine-producing neuron), endoderm(Sox17-positive cells and PDX-positive pancreatic β cells).

FIG. 8 shows the pluripotency (in vitro) of cells obtained by themethods according to the present invention. Teratoma produced bysubcutaneous administration to immunodeficient mice of virus-free iPScells, NHLs and NHL1, induced by the vectors of the present inventionfrom human BJ cells. a: various differentiated tissues. b: cartilage andsecretory cell (black arrow). c: bone tissue. d: secretory tissue (blackarrow) and retina-like tissue (white arrow) differentiated from theneuroepithelium. e: transitional epithelial tissue (center). f:cartilage and myeloid tissue (white arrow). g: gastrointestinal-liketissue. h: spherical tissue. i: myocardial-like tissue

FIG. 9 shows the epigenetics of the cells obtained by the methodsaccording to the present invention. a: The activated state of the humanES cell-specific promoter region was analyzed by the bisulfitesequencing method for each of Oct3/4 and Nanog. The activateddemethylated regions are indicated by white circles and methylatedregions are indicated by black circles. Analyses were carried out onSeV-iPS clones, HNL1 and HNLs, derived from the parent human neonatalforeskin cell line BJ and on SeV-iPS clone 7H5 derived from human adultskin cells HDF. All SeV-iPS cells showed activation of the correspondingpromoters in both regions. b: Gene expression analyses by microarraywere carried out on the virus-free iPS cell HNL1 induced by ScV fromhuman BJ cells, and comparison with BJ cells which are the parent cellline and human ES cell line H9 was performed. The correlationcoefficient is indicated as r. As a result, compared to the alreadyreported retrovirus-induced human iPS cell HDF-iPS, SeV-iPS which becamefree of foreign genes showed a profile closer to that of human ES cellline H9 (correlation coefficient r=0.9789).

FIG. 10 shows elimination of the introduced foreign genes and Sendaivirus (SeV) vectors through cell proliferation. This is a figure showingthat the introduced foreign genes and Sendai virus (SeV) vectors becomediluted/eliminated as the cells proliferate. (A) Decrease in expressionof the introduced foreign genes in SeV-iPS cells derived from neonatalcells (BJ) or from adult cells (HDF) (BJ-derived clones using 18+c-Myc:4BJ1, B1; HDF-derived clone: 7H5; BJ-derived clones using HNL-c-Myc:HNLs, HNL1 to 6, HNLp) were measured over time by RT-PCR using primersrecognizing the sequence of the vector portion (P refers to the passagenumber). As the passage progressed, the four introduced reprogrammingfactors decreased to three and then to two. The tendency observed wasthat when the foreign gene was introduced to position 18+, c-Myc wasdeleted first, and when c-Myc was introduced to position HNL, c-Mycremained until the end, and combinations among the four vectors weresuggested to have certain replicative advantages. Furthermore, clonesHNLs and HNL1 induced with HNL-c-Myc were completely free of foreigngenes. (B) Decrease of the SeV genome in iPS cells over time. In amanner similar to A, decrease in the SeV genome over time in each of theiPS cell clones was measured by quantitative RT-PCR. As a result,decrease of the SeV genome as the passage progressed was confirmed byquantitative PCR as well, and disappearance of the SeV genome in theHNL1 and HNLs clones was made clear. (C) Elimination of the SeV proteinin iPS cells. Elimination of the SeV-derived gene in HNL1 and HNLsobserved in A and B was observed in Western blotting using anti-SeVantibodies as well, and not only the genome but also the SeV-derivedprotein was confirmed to be eliminated.

FIG. 11 shows collection of virus vector-negative cell population usinganti-virus protein antibodies. (Top panels) Virus vector dilution in theiPS colonies is shown. Staining of SeV-iPS cell colonies using anti-HNantibodies suggested that SeV-positive cells and SeV-negative cellscoexist in the colonies, and as shown in the schematic diagram on theleft, by selecting a portion with few virus particles, negativepopulation can be collected (P: passage number). (Lower panels) It wasactually possible to remove SeV-positive cells using anti-HN antibodiesby using HN antigens that appear on the surface of SeV-infected cells asindicators. The SeV-iPS cell population (cell line in which c-Myc/SeVremains: NHLp4 parent) was reacted with the anti-HN antibodies, this wasbound to IMag (BD) magnet beads, the negative fraction was collected,and RT-PCR was used to confirm that this is SeV negative (HNL4p−). Itwas also possible to concentrate the SeV-positive population (HNL4p±).

FIG. 12 shows the properties of the new temperature-sensitive strains.The TS strain used in Example 1 has low cytotoxicity at 37° C., butexpression of the carried GFP protein showed relatively little changeeven when the temperature was shifted from 35° C. to 39° C. (controlTS/ΔF). However, the newly constructed TS7 (Y942H, L1361C, and L1558I)did not show GIP expression at 38° C. or higher, TS13 (P2, L1558I)showed lower expression at 37° C. than at 35° C., and TS15 (P2, L1361C,L1558I) showed hardly any GFP expression at 37° C.

FIG. 13 shows human iPS cell induction using the temperature-sensitivestrains TS7, TS13, and TS15/ΔF/SeV and virus removal. A. ΔF/TS/SeVcarrying c-Myc at the HNL position (between the HN gene and the L gene)of TS7, TS13, or TS15, and ΔF/TS/SeV carrying Oct3/4, Sox2, and KLF4installed in TS were infected simultaneously into neonatal foreskincells BJ to induce human iPS cells. A. Half or more of the isolated iPScells became free of foreign genes as a result of RT-PCR. B. When theSeV protein in the iPS cell clones free of foreign genes was checked,the clones were completely virus-free at the protein level as well.(4BJ1 and B1: SeV-expressing iPS cells; control: SeV-infected LLC-MK2cells).

FIG. 14 shows ES marker expression of human iPS cells induced bytemperature-sensitive strains TS7, TS13, and TS15/ΔF/SeV. Expression ofES markers was verified by RT-PCR for iPS cell clones confirmed to beforeign genes and virus-free in the experiment indicated in FIG. 13. Inall of the virus-free iPS cells, expression of all of the investigatedES markers was confirmed.

FIG. 15 shows SeV-iPS cells induced by other SeV vectors orreprogramming factors. (A) When iPS cell induction was performed byinstalling Oct3/4, Sox2, Oct4, and Nanog onto Lm(Y1214F) ΔF/SeV, whichhas a different vector backbone from TS ΔF/SeV used in Example 1,alkaline phosphatase (ALP)-positive ES-like cell colonies were obtained.(B) iPS cell induction by the four Thomson factors (Oct3/4, Sox2, Nanog,Lin28 ΔF/TS/SeV). iPS cells were induced by factors other than the fourYamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) (left panel), which are thefour Thomson factors (Oct3/4, Sox2, Nanog, Lin28 ΔF/TS/SeV) (rightpanel).

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the mode for carrying out the present invention will bedescribed in detail.

The present invention provides methods for inducing reprogramming ofdifferentiated cells using a chromosomally non-integrating-type virusvector, in particular, methods for producing pluripotent stem cells fromsomatic cells. The methods comprise the step of contacting achromosomally non-integrating virus vector carrying a gene encoding, forexample, a nuclear reprogramming factor to be introduced withdifferentiated cells such as somatic cells. More specifically, thepresent invention provides methods for introducing genes in thereprogramming of cells, in which the genes are introduced using achromosomally non-integrating virus vector into cells in need thereof,and compositions containing a chromosomally non-integrating virus vectorfor that purpose. In the present invention, pluripotent stem cells referto stem cells produced from the inner cell mass of an embryo of ananimal in the blastocyst stage or cells having phenotypes similar tothose cells. Specifically, pluripotent stem cells induced in the presentinvention are cells that express alkaline phosphatase which is anindicator of ES-like cells. Furthermore, preferably, when pluripotentstem cells are cultured, they form flat colonies containing cells with ahigher proportion of nucleus than cytoplasm. Culturing can be carriedout suitably with a feeder. Moreover, while cultured cells such as MEFstop proliferating in a few weeks, pluripotent stem cells can bepassaged for a long period of time, and this can be confirmed based ontheir proliferative character that is not lost even when they arepassaged, for example, 15 times or more, preferably 20 times or more, 25times or more, 30 times or more, 35 times or more, or 40 times or moreevery three days. Furthermore, pluripotent stem cells preferably expressendogenous Oct3/4 or Nanog, or more preferably, they express both ofthem. Furthermore, pluripotent stem cells preferably express TERT, andshow telomerase activity (activity to synthesize telomeric repeatsequences). Moreover, pluripotent stem cells preferably have the abilityto differentiate into three germ layers (the endoderm, mesoderm, andectoderm) (for example, during teratoma formation and/or embryoid bodyformation). More preferably, pluripotent stem cells produce germlinechimera when they are transplanted into blastocysts. Pluripotent stemcells capable of germline transmission are called germline-competentpluripotent stem cells. Confirmation of these phenotypes can be carriedout by known methods (WO 2007/69666; Ichisaka T. et al., Nature 448(7151):313-7, 2007).

Furthermore, in the present invention, “differentiated” refers to, forexample, being more differentiated as compared to pluripotent stemcells, and includes states still possessing the ability to differentiateinto multiple cell lineages (for example, somatic stem cells) andterminally differentiated states. Differentiated cells are cells (otherthan pluripotent stem cells) derived from pluripotent stem cells.Differentiated cells may be, for example, cells that do not have theability to differentiate into the three germ layers (the endoderm,mesoderm, and ectoderm). Such cells will not have the ability to formthe three germ layers unless they are reprogrammed. Furthermore,differentiated cells may be, for example, cells that cannot producecells that are not of the germ layer type to which they belong.Differentiated cells may be somatic cells, and for example, they may becells other than germ cells.

In the present invention, reprogramming refers to converting thedifferentiation state of a particular cell to a less differentiatedstate, and includes for example, dedifferentiation of differentiatedcells, such as inducing cells with differentiation pluripotency, forexample pluripotent stem cells, from cells without differentiationpluripotency. Furthermore, in the present invention, dedifferentiationrefers to converting a particular cell into a more premature (forexample, undifferentiated) state. Dedifferentiation may be reverting acell to its initial state or intermediate state in its path ofdifferentiation. Furthermore, dedifferentiation may be a change from acell unable to produce cells that are not of the same germ layer type,into a cell that can differentiate into other germ layer type cells.Dedifferentiation also includes, for example, cells not havingtriploblastic differentiation ability acquiring this triploblasticdifferentiation ability. Additionally, dedifferentiation includes theproduction of pluripotent stem cells.

Furthermore, in the present invention, somatic cells are, for example,cells other than pluripotent stem cells. Somatic cells include, forexample, multicellular organism-constituting cells other thanpluripotent stem cells, and cultured cells thereof. Somatic cellsinclude for example, somatic stem cells and terminally differentiatedcells.

In the present invention, virus vectors are vectors having genomicnucleic acids derived from the virus, and that can express transgenes byintegrating the transgenes into the nucleic acids. Furthermore,chromosomally non-integrating virus vectors for producing pluripotentstem cells in the present description are virus vectors derived fromviruses and which can introduce genes into target cells, and refer tocarriers that do not involve the risk of having the introduced geneintegrated into the chromosome (nucleus-derived chromosome) of the host.By constructing chromosomally non-integrating virus vectors such asthose that harbor foreign genes, recombinant non-integrating virusvectors used in the present invention can be obtained. Furthermore, inthe present invention, virus vectors include infecting virus particles,as well as complexes of the viral core, viral genome, and viral proteinsand complexes containing non-infectious viral particles and such, whichare complexes having the ability to express loaded genes uponintroduction into cells. For example, in RNA viruses, ribonucleoproteinscontaining a viral genome and viral proteins that bind to it (the viralcore portion) can express transgenes in cells when they are introducedinto cells (WO00/70055). Introduction into cells can be carried outusing appropriate transfection reagents and the like. Suchribonucleoproteins (RNPs) are also included in the virus vectors of thepresent invention.

In the present invention, “no risk of integration into the hostchromosome” indicates that the frequency of integration into the hostchromosome, when the viral vector is introduced, is sufficiently low.Preferably, the frequency of integration into a host chromosome is, forexample, 5×10⁴ or less, more preferably 10⁻⁴ or less, more preferably10⁻⁵ or less, more preferably 10⁻⁶ or less, or more preferably 10⁻⁷ orless when infecting human fibrosarcoma-derived cell line HT1080 (ATCCCCL121) at 10 PFU/cell. The non-integrating virus vectors used in thepresent invention are particularly preferably RNA viruses. In thepresent invention, RNA viruses refer to viruses having an RNA genome,and not having a DNA phase during their lifecycle. In the presentinvention, RNA viruses do not carry reverse transcriptases (that is,retroviruses are not included). Thus, in viral proliferation, the viralgenome is replicated by RNA-dependent RNA polymerases without themediation of DNA. Since RNA viruses do not have a DNA phase, the use ofRNA virus vectors helps to keep the risk of integration into the hostchromosome at a minimum. RNA viruses include single-stranded RNA viruses(including plus strand RNA viruses and minus-strand RNA viruses) anddouble-stranded RNA viruses. Furthermore, they include viruses withenvelope (enveloped viruses) and viruses without envelopes(non-enveloped viruses), but preferably, vectors derived from envelopedviruses are used. In the present invention, RNA viruses specificallyinclude viruses belonging to the following families:

Arenaviridae family such as Lassa virus;

Orthomyxoviridae family such as influenza virus;

Coronaviridae family such as SARS virus;

Togaviridae family such as rubella virus;

Paramyxoviridae family such as mumps virus, measles virus, Sendai virus,and RS virus;

Picornaviridae family such as poliovirus, Coxsackie virus, andechovirus;

Filoviridae family such as Marburg virus and Ebola virus;

Flaviviridae family such as yellow fever virus, dengue fever virus,hepatitis C virus, and hepatitis G virus;

Bunyaviridae family (including the genera Bunyavirus, Hantavirus,Nairovirus, and Phlebovirus);

Rhabdoviridae family such as rabies virus; and

Reoviridae family.

Examples of chromosomally non-integrating virus vectors used in thepresent invention include minus-strand RNA virus vectors. Minus-strandRNA virus vectors are vectors consisting of a virus containing a minusstrand (an antisense strand of a viral protein-encoding sense strand)RNA as the genome. A minus-strand RNA is also referred to as anegative-strand RNA. The minus-strand RNA viruses presented as examplesin the present invention particularly include single-strandedminus-strand RNA viruses (also referred to as non-segmented minus-strandRNA viruses). “Single-stranded negative-strand RNA virus” refers to avirus having a single-stranded negative-strand (i.e., minus-strand) RNAas genome. Such viruses include viruses belonging to families such asParamyxoviridae (including the genera Paramyxovirus, Morbillivirus,Rubulavirus, and Pneumovirus), Rhabdoviridae (including the generaVesiculovirus, Lyssavirus, and Ephemerovirus), and Filoviridae, andtaxonomically belong to Mononegavirales (Virus vol. 57, no. 1, pp.29-36, 2007; Annu. Rev. Genet. 32, 123-162, 1998; Fields virology fourthedition, Philadelphia, Lippincott-Raven, 1305-1340, 2001; Microbiol.Immunol. 43, 613-624, 1999; Field Virology, Third edition pp. 1205-1241,1996).

Minus-strand RNA virus vectors exemplified in the present inventioninclude paramyxovirus vectors. Paramyxovirus vector is a virus vectorderived from a Paramyxoviridae family virus. Examples of aParamyxoviridae virus include Sendai virus. Other examples includeNewcastle disease virus, mumps virus, measles virus, respiratorysyncytial virus (RS virus), rinderpest virus, distemper virus, simianparainfluenza virus (SV5), and human parainfluenza viruses I, II, andIII; influenza virus belonging to the Orthomyxoviridae family; and thevesicular stomatitis virus and Rabies virus belonging to theRhabdoviridae family.

Further examples of viruses that may be used in the present inventioninclude Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), humanparainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), caninedistemper virus (CDV), dolphin molbillivirus (DMV),peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpestvirus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), humanparainfluenza virus-2 (HPIV-2), simian parainfluenza virus 5 (SV5),human parainfluenza virus-4a (HPIV-4a), human parainfluenza virus-4b(HPIV-4b), mumps virus (Mumps), and Newcastle disease virus (NDV). Morepreferably, examples include viruses selected from the group consistingof Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), humanparainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), caninedistemper virus (CDV), dolphin molbillivirus (DMV),peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpestvirus (RPV), Hendra virus (Hendra), and Nipah virus (Nipah).

Vectors used in the present invention are, for example, virusesbelonging to the Paramyxoviridae subfamily (including the generarespirovirus, rubulavirus, and morbillivirus) or derivatives thereof,and examples include viruses belonging to the genus Respirovirus (alsoreferred to as the genus Paramyxovirus) or derivatives thereof.Derivatives include chemically modified viruses and viruses whose viralgenes have been modified such that the gene transfer ability of thevirus is not impaired. Examples of Respirovirus viruses to which thepresent invention can be applied include human parainfluenza virus 1(HPIV-1), human parainfluenza virus 3 (HPIV-3), bovine parainfluenzavirus 3 (BPIV-3), Sendai virus (also called mouse parainfluenza virus1), and simian parainfluenza virus 10 (SPIV-10).

Minus strand RNA viruses exemplified in the present invention morespecifically include Sendai viruses. The genome of wild-type Sendaivirus includes a short 3′ leader region followed by a nucleocapsid (N)gene, a phospho (P) gene, a matrix (M) gene, a fusion (F) gene, ahemagglutinin-neuraminidase (HN) gene, and a large (L) gene, and then ashort 5′ trailer region, in this order. Production of recombinantvectors corresponding to wild-type viruses, and of various mutantvectors are already known. Furthermore, it has been shown that genetransfer is possible using the RNP alone without its envelope(WO00/70055). Therefore, reprogramming using RNP is also included in thepresent invention. The same is true with other viral RNPs.

Chromosomally non-integrating viruses in the present invention may bederived from natural strains, wild-type strains, mutant strains,laboratory-passaged strains, artificially constructed strains, and such.That is, these viruses may be virus vectors having similar structures asviruses isolated from nature, or viruses artificially modified bygenetic recombination, as long as the desired reprogramming can beinduced. For example, they may have mutations or deletions in any of thegenes of the wild-type virus. Furthermore, incomplete viruses such as DIparticles (J. Virol. 68: 8413-8417, 1994) may also be used. For example,viruses having a mutation or deletion in at least one gene encoding aviral envelope protein or a coat protein can be suitably used. Suchvirus vectors are, for example, virus vectors that can replicate thegenome in infected cells but cannot form infectious virus particles.Since there is no worry of spreading the infection to the surroundings,such replication-defective virus vectors are very safe. For example,minus-strand RNA viruses that do not contain at least one gene encodingan envelope protein such as F, H, HN, or G, or a spike protein, or acombination thereof may be used (WO00/70055 and WO00/70070; Li, H.-O. etal., J. Virol. 74(14) 6564-6569 (2000)). If proteins necessary forgenome replication (for example, N, P, and L proteins) are encoded inthe genomic RNA, the genome can be amplified in infected cells. Toproduce defective type of viruses, for example, the defective geneproduct or a protein that can complement it is externally supplied inthe virus-producing cell (WO00/70055 and WO00/70070; Li, H.-O. et al.,J. Virol. 74(14) 6564-6569 (2000)). Furthermore, a method of collectingvirus vectors as noninfective virus particles (VLP) without completelycomplementing the defective viral protein is also known (WO00/70070).Furthermore, when virus vectors are collected as RNPs (for example, RNPscontaining the N, L, and P proteins and genomic RNA), vectors can beproduced without complementing the envelope proteins.

Furthermore, the use of virus vectors carrying a mutant viral proteingene is also preferred. The present invention particularly providesmethods of gene transfer in reprogramming and methods for producingreprogrammed cells using RNA virus vectors having mutations and/ordeletions in the viral gene. For example, in the envelope protein andcoat proteins, many mutations including attenuation mutations andtemperature-sensitive mutations are known. RNA viruses having thesemutant protein genes can be used favorably in the present invention. Inthe present invention, vectors with lowered cytotoxicity are desirablyused. Cytotoxicity can be measured, for example by quantifying therelease of lactic acid dehydrogenase (LDH) from cells. For example,vectors with significantly lowered cytotoxicity compared to the wildtype can be used. Regarding the degree of lowering of cytotoxicity, forexample, vectors showing a significant decrease of, for example 20% ormore, 25% or more, 30% or more, 35% or more, 40% or more, or 50% or morein the LDH release level compared to the wild-type in a culture mediumof HeLa (ATCC CCL-2) or simian CV-1 (ATCC CCL 70) infected at MOI 3 andcultured for three days can be used. Furthermore, mutations thatdecrease cytotoxicity also include temperature-sensitive mutations.Temperature-sensitive mutations refer to mutations which significantlydecrease the activity at the viral host's ordinary temperature (forexample, 37° C. to 38° C.) when compared to that at a low temperature(for example, 30° C. to 32° C.). Such proteins withtemperature-sensitive mutations are useful since the viruses can beproduced under permissive temperatures (low temperatures). When infectedat 37° C., the virus vectors having useful temperature-sensitivemutations in the present invention show, a growth rate or geneexpression level of, for example, ½ or less, preferably ⅓ or less, morepreferably ⅕ or less, more preferably 1/10 or less, and more preferably1/20 or less compared to when cultured cells are infected at 30° C.

A chromosomally non-integrating virus vector used in the presentinvention may be a wild type as long as it does not inhibitreprogramming and can induce reprogramming by reprogramming factors, andhas deletions or mutations in preferably at least one, more preferablyat least 2, 3, 4, 5, or more viral genes. Deletions and mutations may bearbitrarily combined and introduced to each of the genes. Herein, amutation may be a function-impairing mutation or a temperature-sensitivemutation, and is a mutation that decreases the viral proliferation rateor the expression level of the carried gene to preferably ½ or less,more preferably ⅓ or less, more preferably ⅕ or less, more preferably1/10 or less, and more preferably 1/20 or less compared to the wild typeat least at 37° C. The use of such modified virus vectors can beimportant particularly for the induction of pluripotent stem cells. Forexample, minus-strand RNA virus vectors used favorably in the presentinvention have at least two deleted or mutated viral genes. Such virusesinclude those with deletions of at least two viral genes, those withmutations in at least two viral genes, and those with a mutation in atleast one viral gene and a deletion of at least one viral gene. The atleast two mutated or deleted viral genes are preferably genes encodingenvelope-constituting proteins. For example, vectors with deletion ofthe F gene with further deletion of the M and/or the HN (or H) gene orfurther mutation (for example, temperature-sensitive mutation) in the Mand/or the HN (or H) gene are used favorably in the present invention.Furthermore, for example, vectors with deletion of the F gene withfurther deletion of the M or the HN (or H) gene and further mutation inthe remaining M and/or the HN (or H) gene (for example,temperature-sensitive mutation) are also used favorably in the presentinvention. Vectors used in the present invention more preferably have atleast three deleted or mutated viral genes (preferably at least threegenes encoding envelope-constituting proteins). Such virus vectorsinclude those with deletion of at least three genes, those withmutations in at least three genes, those with mutations in at least onegene and deletion of at least two genes, and those with mutations in atleast two genes and deletion of at least one gene. As examples of morepreferred embodiments, vectors with deletion of the F gene with furtherdeletion of the M and the HN (or H) gene or further mutations (forexample, temperature-sensitive mutations) in the M and the HN (or H)gene are used favorably in the present invention. Furthermore, forexample, vectors with deletion of the F gene with further deletion ofthe M or the HN (or H) gene and further mutation in the remaining M orthe HN (or H) gene (for example, temperature-sensitive mutation) arealso used favorably in the present invention. Such mutated-form virusescan be produced according to known methods.

For example, a temperature-sensitive mutation of the M gene of theminus-strand RNA virus includes amino acid substitution of a sitearbitrarily selected from the group consisting of position 69 (G69),position 116 (T116), and position 183 (A183) of the M protein of aSendai virus or a homologous site of another minus-strand RNA virus Mprotein (Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Amino acidsof homologous sites in the M protein of other minus strand RNA virusescan be identified easily, but specifically, the homologous site in an Mprotein corresponding to G69 in the SeV M protein include G69 for humanparainfluenza virus-1 (HPIV-1) (abbreviation is indicated inparenthesis), G73 for human parainfluenza virus-3 (HPIV-3), G70 forphocine distemper virus (PDV) and canine distemper virus (CDV), G71 fordolphin molbillivirus (DMV), G70 for peste-des-petits-ruminants virus(PDPR), measles virus (MV), and rinderpest virus (RPV), G81 for Hendravirus (Hendra) and Nipah virus (Nipah), G70 for human parainfluenzavirus-2 (HPIV-2), E47 for human parainfluenza virus-4a (HPIV-4a) andhuman parainfluenza virus-4b (HPIV-4b), and E72 for mumps virus (Mumps)(the letter and number indicate the amino acid and its position). Thehomologous sites in each of the M proteins corresponding to T116 of theSeV M protein include T116 for human parainfluenza virus-1 (HPIV-1),T120 for human parainfluenza virus-3 (HPIV-3), T104 for phocinedistemper virus (PDV) and canine distemper virus (CDV), T105 for dolphinmolbillivirus (DMV), T104 for peste-des-petits-ruminants virus (PDPR),measles virus (MV), and rinderpest virus (RPV), T120 for Hendra virus(Hendra) and Nipah virus (Nipah), T117 for human parainfluenza virus-2(HPIV-2) and simian parainfluenza virus 5 (SV5), T121 for humanparainfluenza virus-4a (HPIV-4a) and human parainfluenza virus-4b(HPIV-4b), T119 for mumps virus (Mumps), and S120 for Newcastle diseasevirus (NDV). The homologous sites in each of the M proteinscorresponding to A183 of the SeV M protein include A183 for humanparainfluenza virus-1 (HPIV-1), F187 for human parainfluenza virus-3(HPIV-3), Y171 for phocine distemper virus (PDV) and canine distempervirus (CDV), Y172 for dolphin molbillivirus (DMV), Y171 forpeste-des-petits-ruminants virus (PDPR), measles virus (MV), andrinderpest virus (RPV), Y187 for Hendra virus (Hendra) and Nipah virus(Nipah), Y184 for human parainfluenza virus-2 (HPIV-2), F184 for simianparainfluenza virus 5 (SV5), F188 for human parainfluenza virus-4a(HPIV-4a) and human parainfluenza virus-4b (HPIV-4b), F186 for mumpsvirus (Mumps), and Y187 for Newcastle disease virus (NDV). Among theviruses mentioned above, viruses having a genome encoding a mutant Mprotein, in which the amino acids of any one site, preferably acombination of any two sites, or more preferably all three sites of thethree sites mentioned above are substituted in the respective M proteinsto other amino acids, are used preferably in the present invention.

Preferred amino acid mutations are substitution to other amino acidswith a side chain having different chemical properties, and examples aresubstitution to an amino acid with a BLOSUM62 matrix (Henikoff, S. andHenikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) scoreof three or less, preferably two or less, more preferably one or less,and even more preferably 0 or less. Specifically, G69, T116, and A183 ofthe Sendai virus M protein or homologous sites in the M protein of otherviruses can be substituted to Glu (E), Ala (A), and Ser (S),respectively. Alternatively mutations homologous to mutations in the Mprotein of the temperature-sensitive P253-505 measles virus strain(Morikawa, Y. et al., Kitasato Arch. Exp. Med. 1991: 64; 15-30) can alsobe used. Mutations can be introduced according to known mutationmethods, for example, using oligonucleotides and such.

Furthermore, examples of temperature-sensitive mutations in the HN (orII) gene include amino acid substitution of a site arbitrarily selectedfrom the group consisting of position 262 (A262), position 264 (G264),and position 461 (K461) of the HN protein of a Sendai virus or ahomologous site in the M protein of other minus-strand RNA viruses(Inoue, M. et al., J. Virol. 2003, 77: 3238-3246). Viruses having agenome encoding a mutant HN protein in which the amino acids of any oneof the three sites, preferably a combination of any two sites, or morepreferably all three sites are substituted to other amino acids are usedpreferably in the present invention. As mentioned above, preferred aminoacid substitutions are substitution to other amino acids with a sidechain having different chemical properties. As a preferred example,A262, G264, and K461 of the Sendai virus HN protein or homologous sitesin the HN protein of other viruses can be substituted to Thr (T), Arg(R), and Gly (G), respectively. Furthermore, for example, using thetemperature-sensitive vaccine strain Urabe AM9 of the mumps virus as areference, amino acids of positions 464 and 468 of the HN protein can bemutated (Wright, K. E. et al., Virus Res. 2000: 67; 49-57).

Furthermore, minus-strand RNA viruses may have mutations in the P geneand/or the L gene. Examples of such mutations are specifically, mutationof Glu at position 86 (E86) in the SeV P protein, and substitution ofLeu at position 511 (L511) in the SeV P protein to other amino acids, orsubstitution of homologous sites in the P protein of other minus-strandRNA viruses. As mentioned above, preferred amino acid substitutions aresubstitutions to other amino acids with a side chain having differentchemical properties. Specific examples include substitution of the aminoacid at position 86 to Lys, and substitution of the amino acid atposition 511 to Phe. Furthermore, examples in the L protein includesubstitution of Asn at position 1197 (N1197) and/or Lys at position 1795(K1795) in the SeV L protein to other amino acids, or substitutions ofhomologous sites in the L protein of other minus-strand RNA viruses, andsimilarly as above, preferred amino acid substitutions are substitutionsto other amino acids with a side chain having different chemicalproperties. Specific examples are substitution of the amino acid atposition 1197 to Ser, and substitution of the amino acid at position1795 to Glu. Mutations of the P gene and L gene can significantlyincrease the effects of sustained infectivity, suppression of release ofsecondary particles, or suppression of cytotoxicity. Further,combination of mutations and/or deletions of envelope protein genes candramatically increase these effects. Furthermore, examples for the Lgene include substitution of Tyr at position 1214 (Y1214) and/orsubstitution of Met at position 1602 (M1602) of the SeV L protein toother amino acids, or substitution of homologous sites in the L proteinof other minus-strand RNA viruses, and similarly as above, preferredamino acid substitutions are substitutions to other amino acids with aside chain having different chemical properties. Specific examples aresubstitution of the amino acid at position 1214 to Phe, and substitutionof the amino acid at position 1602 to Leu. The above-mentioned mutationscan be arbitrarily combined.

For example, Sendai virus vectors in which at least G at position 69, Tat position 116, and A at position 183 of the SeV M protein, at least Aof position 262, G of position 264, and K of position 461 of the SeV HNprotein, at least L of position 511 of the SeV P protein, and at least Nof position 1197 and K of position 1795 of the SeV L protein are eachsubstituted to other amino acids, and in which the F gene is alsodeficient or deleted; F-gene-deleted or -deficient vectors havingsubstitution mutations at homologous sites in each of the homologousproteins of other minus-strand RNA viruses and having a deleted ordeficient F gene; and F-gene-deleted or -deficient minus-strand RNAvirus vectors whose cytotoxicity is similar to or lower than thosementioned above and/or whose temperature sensitivity is similar to orhigher than those mentioned above are particularly preferred for theexpression of nuclear reprogramming factors in the present invention.Specific examples of the substitutions include G69E, T116A, and A183Ssubstitutions for the M protein, A262T, G264, and K461G substitutionsfor the HN protein, L511F substitution for the P protein, and N1197S andK1795E substitutions for the L protein. Genes encoding nuclearreprogramming factors can be positioned, for example, at the mostupstream position (3′ side) of the minus-strand RNA genome (for example,at the 3′ side of the N gene). However, regarding the Myc gene, it maybe positioned at other positions, for example, at the rear of theminus-strand RNA genome, that is, more towards the 5′ side. For example,it may be inserted between the HN gene and the L gene.

Examples of mutations of the L protein include substitutions of an aminoacids at sites arbitrarily selected from position 942 (Y942), position1361 (L1361), and position 1558 (L1558) of the SeV L protein to otheramino acids, or substitutions of homologous sites in the L protein ofother minus-strand RNA viruses. Similarly as above, preferred amino acidsubstitutions are substitution to other amino acids with a side chainhaving different chemical properties. Specific examples includesubstitution of the amino acid of position 942 to His, substitution ofthe amino acid of position 1361 to Cys, and substitution of the aminoacid of position 1558 to Ile. In particular, the L protein withsubstitutions at least at positions 942 and 1558 can be used preferably.For example, mutant L proteins in which, in addition to position 1558,position 1361 is also substituted to another amino acid are preferred aswell. Furthermore, mutant L proteins in which, in addition to position942, position 1558 and/or position 1361 are also substituted to otheramino acids are favorable as well. Mutant L proteins with mutations toother amino acids at position 1558 and/or position 1361 in addition toposition 942 are also preferred. These mutations can increase thetemperature sensitivity of the L protein.

Examples of mutations of the P protein include substitutions of aminoacids at sites arbitrarily selected from position 433 (D433), position434 (R434), and position 437 (K437) of the SeV P protein to other aminoacids, or substitutions of homologous sites in the P protein of otherminus-strand RNA viruses. Similarly as above, preferred amino acidsubstitutions are substitution to other amino acids with a side chainhaving different chemical properties. Specific examples includesubstitution of the amino acid of position 433 to Ala (A), substitutionof the amino acid of position 434 to Ala (A), and substitution of theamino acid of position 437 to Ala (A). In particular, P proteins inwhich all three of these sites are substituted can be used preferably.These mutations can increase the temperature sensitivity of the Pprotein.

F-gene-deleted or -deficient Sendai virus vectors encoding a mutant Pprotein in which at least at the three positions of D at position 433, Rat position 434, and K at position 437 of the SeV P protein aresubstituted to other amino acids, and a mutant L protein in which atleast the L at position 1558 of the SeV L protein is substituted(preferably a mutant L protein in which at least the L at position 1361is also substituted to another amino acid); and F-gene-deleted or-deficient vector in which homologous sites in other minus-strand RNAviruses are mutated; and F-gene-deleted or -deficient minus-strand RNAvirus vectors whose cytotoxicity is similar to or lower than thosementioned above and/or whose temperature sensitivity is similar to orhigher than those mentioned above are used preferably in the presentinvention. In addition to the above-mentioned mutations, each of theviral proteins may have mutations on other amino acids (for example, onten or less, five or less, four or less, three or less, two or less, orone amino acid). Since vectors comprising the above-mentioned mutationsshow a high temperature sensitivity, after completion of reprogramming,the vectors can be removed easily by culturing the cells at a slightlyhigh temperature (for example, 37.5° C. to 39° C., preferably 38° C. to39° C., or 38.5° C. to 39° C.). Nuclear reprogramming factors can beinserted into appropriate sites of a suitable genome, and for example,they are inserted at the most upstream position (3′ side) of the genome(for example, at the 3′ side of the NP gene). Regarding the Myc gene, itmay be positioned, for example, at the 5′ end side from the center ofthe minus-strand RNA virus genome (at the 5′ end side from the gene atthe center), for example, it may be inserted at the 5′ side or the 3′side of the L gene, and particularly at the 3′ side of the L gene (forexample between HN and L).

The cytotoxicity of vectors can be measured, for example, by quantifyingthe release of lactate dehydrogenase (LDH) from cells. Specifically, forexample, HeLa (ATCC CCL-2) or simian CV-1 (ATCC CCL70) is infected atMOI 3, and the amount of LDH released into the culture solution afterthree days of culture is measured. The lower the amount of LDH released,the lower the cytotoxicity. Furthermore, temperature sensitivity can bedetermined by measuring the speed of viral proliferation or theexpression level of the installed gene at the viral host's ordinarytemperature (for example, 37° C. to 38° C.). The lower the speed ofviral proliferation and/or expression level of the installed gene ascompared to those without mutations, the higher the temperaturesensitivity is judged to be.

Furthermore, when using an envelope virus, a virus containing a proteinin the envelope that is different from the envelope protein originallycarried by the virus may be used. For example, by expressing a desiredexogenous envelope protein in a virus-producing cell when producing thevirus, a virus containing this protein can be produced. Such proteinsare not particularly limited, and desired proteins, such as adhesionfactors, ligands, and receptors, that confer mammalian cells with aninfectious ability are used. Specific examples include the G protein ofVesicular stomatitis virus (VSV) (VSV-G). The VSV-G protein may bederived from any VSV strain, and for example, VSV-G protein derived fromthe Indiana serotype strain (J. Virology 39: 519-528 (1981)) may beused, but it is not limited thereto. The minus-strand RNA virus given asan example in the present invention can include arbitrary combinationsof other virus-derived envelope proteins.

Reconstitution of recombinant RNA viruses carrying nuclear reprogrammingfactors can be carried out using well-known methods. As specificprocedures, typically, the minus-strand RNA viruses cited as an examplein the present invention can be produced by the steps of (a)transcribing a cDNA encoding the minus-strand RNA virus genomic RNA(minus strand) or a complementary strand thereof (plus strand) in a cellthat expresses viral proteins (N, P, and L) necessary for virus particleformation, and (b) collecting a culture supernatant containing theproduced viruses. Viral proteins necessary for particle formation may beexpressed from the transcribed viral genomic RNA, or they may beprovided in trans from sources other than genomic RNA. For example, theycan be provided by introducing expression plasmids encoding the N, P,and L proteins into cells. When viral genes necessary for particleformation are lacking in the genomic RNA, those viral genes areseparately expressed in virus-producing cells to complement particleformation. To express the viral proteins or the RNA genome in cells,vectors having a DNA encoding such proteins or genomic RNA linkeddownstream of a suitable promoter that functions in a host cell isintroduced into the host cell. The transcribed genomic RNA is replicatedin the presence of viral proteins, and infectious virus particles areformed. When producing a defective type of virus lacking genes such asthose of the envelope proteins, the missing protein, other viralproteins that can complement the function of those proteins, or such areexpressed in the virus-producing cells.

For example, production of the minus-strand RNA viruses exemplified inthe present invention can be carried out by using the following knownmethods (WO97/16539; WO97/16538; WO00/70055; WO00/70070; WO01/18223;WO03/025570; WO2005/071092; WO2006/137517; WO2007/083644; WO2008/007581;Hasan, M. K. et al., J. Gen. Virol. 78: 2813-2820, 1997; Kato, A. etal., 1997, EMBO J. 16: 578-587 and Yu, D. et al., 1997, Genes Cells 2:457-466; Durbin, A. P. et al., 1997, Virology 235: 323-332; Whelan, S.P. et al., 1995, Proc. Natl. Acad. Sci. USA 92: 8388-8392; Schnell. M.J. el al., 1994, EMBO J. 13: 4195-4203; Radecke, F. et al, 1995, EMBO J.14: 5773-5784; Lawson, N. D. et al., Proc. Natl. Acad. Sci. USA 92:4477-4481; Garcin, D. et al., 1995, EMBO J. 14: 6087-6094; Kato, A. elal., 1996, Genes Cells 1: 569-579; Baron, M. D. and Barrett, T., 1997,J. Virol. 71: 1265-1271; Bridgen, A. and Elliott, R. M., 1996, Proc.Natl. Acad. Sci. USA 93: 15400-15404; Tokusumi, T. et al. Virus Res.2002: 86; 33-38; and Li, H.-O. et al., J. Virol. 2000: 74; 6564-6569).Minus-strand RNA viruses including parainfluenza, vesicular stomatitisvirus, rabies virus, measles virus, rinderpest virus, and Sendai viruscan be reconstituted from DNAs by these methods.

Examples of methods for producing plus(+)-strand RNA viruses include thefollowing:

1) Coronavirus

-   Enjuanes L, Sola I, Alonso S, Escors D, Zuniga S.

Coronavirus reverse genetics and development of vectors for geneexpression.

Curr Top Microbiol Immunol. 2005; 287:161-97. Review.

2) Togavirus

-   Yamanaka R, Zullo S A, Ramsey J, Onodera M, Tanaka R, Blaese M,    Xanthopoulos K G

Induction of therapeutic antitumor antiangiogenesis by intratumoralinjection of genetically engineered endostatin-producing Semliki Forestvirus.

Cancer Gene Ther. 2001 October; 8(10):796-802.

-   Datwyler D A, Eppenberger H M, Koller D, Bailey J E, Magyar J P.

Efficient gene delivery into adult cardiomyocytes by recombinant Sindbisvirus.

J Mol Med. 1999 December; 77(12):859-64.

3) Picornavirus

-   Lee S G, Kim D Y, Hyun B H, Bac Y S.

Novel design architecture for genetic stability of recombinantpoliovirus: the manipulation of G/C contents and their distributionpatterns increases the genetic stability of inserts in apoliovirus-based RPS-Vax vector system.

J Virol. 2002 February; 76(4):1649-62.

Mueller S, Wimmer E.

Expression of foreign proteins by poliovirus polyprotein fusion:analysis of genetic stability reveals rapid deletions and formation ofcardioviruslike open reading frames.

J Virol. 1998 January; 72(1):20-31.

4) Flavivirus

-   Yun S I, Kim S Y, Rice C M, Lee Y M.

Development and application of a reverse genetics system for Japaneseencephalitis virus.

J Virol. 2003 June; 77(11):6450-65.

-   Arroyo J, Guirakhoo F, Fenner S, Zhang Z X, Monath T P, Chambers T    J.

Molecular basis for attenuation of neurovirulence of a yellow feverVirus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE).

J Virol. 2001 January; 75(2):934-42.

5) reovirus

-   Roncr M R, Joklik W K.

Reovirus reverse genetics: Incorporation of the CAT gene into thereovirus genome.

Proc Natl Acad Sci USA. 2001 Jul. 3; 98(14):8036-41. Epub 2001 Jun. 26.

Regarding other methods for proliferation of RNA viruses and methods forproducing recombinant viruses, see “Uirusu-gaku Jikken-gaku Kakuron(Detailed Virology Experiments)”, second revised edition (NationalInstitute of Infectious Diseases Students Institute edition, Maruzen,1982).

To the above-mentioned chromosomally non-integrating virus vectors,genes for reprogramming cells can be appropriately installed. The genesto be installed may be desired genes involved in the induction and suchof various stem cells such as pluripotent stem cells from differentiatedcells. For example, such genes necessary for reprogramming or genes thatincrease the efficiency of reprogramming can be installed. Thus, thepresent invention provides uses of the chromosomally non-integratingvirus vectors of the present invention for introducing genes in cellularreprogramming, and uses of these vectors for expressing reprogrammingfactors in cells to induce reprogramming of those cells. Furthermore,the present invention provides agents containing the chromosomallynon-integrating virus vectors of the present invention for introducinggenes in cellular reprogramming (transfer agents, gene transfer agents)and agents containing these vectors for expressing reprogramming factorsin cells. Furthermore, the present invention relates to agentscontaining the chromosomally non-integrating virus vectors of thepresent invention for expressing reprogramming factors in cells toinduce reprogramming of the cells. Furthermore, when carrying outnuclear reprogramming of cells, the vectors of the present invention arealso useful for expressing desired genes in these cells. Non-integratingvirus vectors carrying one or more genes encoding a nuclearreprogramming factor can be utilized for cellular reprogrammingaccording to the present invention. The present invention can be usedfor medical uses and for non-medical uses, and is useful in medical andnon-medical embodiments. For example, the present invention can be usedfor therapeutic, surgical, and/or diagnostic, or non-therapeutic,non-surgical, and/or non-diagnostic purposes.

In the present invention, a nuclear reprogramming factor refers to agene used, by itself or together with a number of factors, for inducinga differentiated state of a certain cell to change to a moreundifferentiated state, or a product thereof, and includes for example,a gene used for inducing dedifferentiation of differentiated cells, or aproduct thereof. The nuclear reprogramming factors in the presentinvention include factors essential for nuclear reprogramming andaccessorial factors (auxiliary factors) which increase the efficiency ofnuclear reprogramming. In the present invention, desired genes to beused for nuclear reprogramming can be installed into a vector. Forexample, genes to be used for the production of pluripotent stem cellscan be installed. Specifically, as the nuclear reprogramming factors forinduction of pluripotent stem cells, for example, genes that areexpressed in ES cells or early embryo but are not expressed or whoseexpression is decreased in many differentiated somatic cells (EScell-specific genes and such) can be used. Such genes are preferablygenes that encode transcription factors, nucleoproteins, or such.Methods for identifying nuclear programming factors are already known(WO2005/80598), and in fact, genes identified using this method havebeen shown to be useful in reprogramming into pluripotent stem cells(WO2007/69666).

Examples of such genes include DPPA5 (developmental pluripotencyassociated 5, ES cell specific gene 1 (ESG1); accession numbersNM_001025290, NM_025274, XM_236761), F-box protein 15 (Fbx15, NM_152676,NM_015798), Nanog (NM_024865, AB093574), ECAT1 (ES cell associatedtranscript 1; AB211062, AB211060), ERAS (ES cell expressed Ras;NM_181532, NM_181548), DNMT3L (DNA (cytosine-5-)-methyltransferase3-like; NM_013369, NM_019448), ECAT8 (A8211063, AB211061), GDF3 (growthdifferentiation factor 3; NM_020634, NM_008108), SOX15 (SRY (sexdetermining region Y)-box 15; NM_006942, NM_009235), DPPA4(developmental pluripotency associated 4; NM_018189, NM_028610), DPPA2(NM_138815, NM_028615), FTHL17 (ferritin, heavy polypeptide-like 17;NM_031894, NM_031261), SALL4 (sal-like 4; NM_020436, NM_175303), Oct3/4(also called POU5F1; NM_002701, NM_203289, NM_013633, NM_001009178),Sox2 (NM_003106, NM_011443, XM_574919), Rex-1 (ZFP42 (zinc fingerprotein 42 homolog); NM_174900, NM_009556), Utf1 (undifferentiatedembryonic cell transcription factor 1; NM_003577, NM_009482), TCL1A(1-cell leukemia/lymphoma 1A; NM_021966, NM_009337), DPPA3 (also calledStella, NM_199286, NM_139218, XM_216263), KLF4 (Kruppel-like factor 4;NM_004235, NM_010637), catenin β1 (cadherin-associated protein beta 1;NM_001904, NM_007614; including the S33Y mutant), c-Myc (NM_002467,NM_010849; including the T58A mutant), STAT3 (signal transducer andactivator of transcription 3; NM_ 139276, NM_213659), GRB2 (growthfactor receptor-bound protein 2; NM_002086, NM_008163), and other geneswhich are members of the families to which these genes belong. Thesegenes have been shown to be able to induce pluripotent stem cells uponintroduction into cells (WO2007/69666). Therefore, a chromosomallynon-integrating virus vector, for example, an RNA virus vector, carryingany one of these genes is useful for use in inducing dedifferentiationof cells in the present invention, and can be used favorably forinduction of pluripotent stem cells in particular. These genes may beincorporated one at a time into separate vectors, or a number of genescan be integrated altogether into a single vector. Furthermore, each ofthe genes may be integrated into a single type of vector, or differenttypes of vectors (including chromosomally integrated virus vectorsand/or non-viral vectors) may be used in combination with chromosomallynon-integrating virus vectors. In addition, individual virus vectors arepackaged separately, and can be used by combining them at the time ofuse. Alternatively, multiple virus vectors carrying different genes canbe combined in advance as a kit, or they may be mixed to produce acomposition. Furthermore, one or more non-integrating virus vectorscontaining any combination (or all) of these genes, and kits orcompositions containing these vectors can be used favorably for cellularreprogramming, particularly in the production of pluripotent stem cells.In the case of compositions, the vectors may be appropriately mixed insterilized water, pH buffers, physiological saline solutions, culturesolutions, and such. In these systems, a part of or most of the nuclearreprogramming genes can be substituted with proteins which are theirexpression products. Thus, the compositions and kits of the presentinvention may include other vectors (chromosomally integrated virusvectors and/or non-viral vectors) that express reprogramming factorsand/or compounds, proteins, or such that induce reprogramming, as longas they include at least one chromosomally non-integrating virus vector.All of the factors necessary for reprogramming may be expressed fromchromosomally non-integrating virus vectors, or only a portion of themmay be expressed from chromosomally non-integrating virus vectors, andthe rest may be provided from other vectors and/or compounds (forexample, proteins or low-molecular weight compounds). Furthermore, themethods of the present invention for producing reprogrammed cells arenot limited to methods in which all gene transfers are carried out usingchromosomally non-integrating virus vectors. More specifically, themethods of the present invention only need to use at least onechromosomally non-integrating virus vector, and includes combined use ofother vectors (chromosomally integrated virus vectors and/or non-viralvectors) expressing reprogramming factors and/or compounds that inducereprogramming and such.

The present invention relates to compositions to be used for cellularreprogramming, which include a chromosomally non-integrating virusvector as the expression vector. Furthermore, the present inventionrelates to use of a chromosomally non-integrating virus vector for usein reprogramming of differentiated cells. For example, the presentinvention provides use of a chromosomally non-integrating virus vectorfor introducing genes for cellular reprogramming into cells in needthereof. Furthermore, the present invention relates to methods forintroducing genes in cellular reprogramming, which use chromosomallynon-integrating virus vectors to introduce genes into cells in needthereof. Furthermore, the present invention also relates to compositionsto be used for gene transfer in cellular reprogramming and agents to beused for gene transfer in cellular reprogramming (transfer agents to beused in gene transfer for cellular reprogramming and gene transferagents for cellular reprogramming), which include a chromosomallynon-integrating virus vector. Furthermore, the present invention relatesto a use of a chromosomally non-integrating virus vector in theproduction of pharmaceutical agents for introducing genes for cellularreprogramming into cells in need thereof. The present invention alsoprovides gene transfer agents (gene expression agents or expressionvectors) for use in cellular reprogramming, which contain chromosomallynon-integrating virus vectors. Furthermore, the present inventionprovides agents for introducing reprogramming genes (gene expressionagents or expression vectors), which contain chromosomallynon-integrating virus vectors. The present invention also provides,agents for expressing nuclear reprogramming factors (nuclearreprogramming gene-transfer agents, nuclear reprogramminggene-expression vectors) which contain chromosomally non-integratingvirus vectors. Furthermore, the present invention provides pluripotentstem cell-inducing agents and pluripotent stem cell-inducing auxiliarygents, which contain chromosomally non-integrating virus vectorsencoding nuclear reprogramming factors. The present invention providesuse of chromosomally non-integrating virus vectors for the reprogrammingof differentiated cells. The present invention also provides use ofchromosomally non-integrating virus vectors in the production ofpharmaceutical agents, reagents, and/or pharmaceuticals for thereprogramming of differentiated cells. The present invention alsorelates to use of chromosomally non-integrating virus vectors in theproduction of agents for introducing nuclear reprogramming factors intodifferentiated cells.

Herein, reprogramming may be, for example, induction of pluripotent stemcells from differentiated cells. Vectors are used by integrating genesencoding factors for reprogramming. Examples of genes encodingreprogramming factors include genes encoding any one of theabove-mentioned factors or factors exemplified below.

The factors that are introduced may be selected appropriately accordingto the origin of the cells to be reprogrammed, and they may be derivedfrom humans or other mammals such as mice, rats, rabbits, pigs, orprimates such as monkeys. Furthermore, the genetic and protein sequencesdo not necessarily have to be wild-type sequences, and as long as theycan induce reprogramming, they may have any mutations. In fact, examplesof producing pluripotent stem cells using mutant genes are known(WO2007/69666). For example, a gene encoding an amino acid sequence withone or a small number of (for example, a few, not more than three, notmore than five, not more than ten, not more than 15, not more than 20,or not more than 25) amino acid additions, deletions, substitutions,and/or insertions, and which can induce reprogramming may be used in thepresent invention. Furthermore, as long as biological activity (abilityto induce reprogramming) is maintained, for example, polypeptides withdeletions or additions of one to several residues (for example, 2, 3, 4,5, 6, 10, 15, or 20 residues) of amino acids of the N terminus and/orthe C terminus, polypeptides with substitution of one to severalresidues (for example, 2, 3, 4, 5, 6, 10, 15, or 20 residues) of aminoacids, and such may be used. Variants which may be used include forexample, fragments, analogs, and derivatives of naturally-derivedproteins, and fusion proteins of naturally derived proteins with otherpolypeptides (for example, those with addition of heterologous signalpeptides or antibody fragments). Specifically, polypeptides comprising asequence with one or more amino acid substitutions deletions, and/oradditions in the wild-type amino acid sequence, and having a biologicalactivity (for example, activity to induce reprogramming) equivalent tothat of wild-type proteins are included. When using a fragment of awild-type protein, normally, the fragment contains a continuous regionof 70% or more, preferably 80% or more, 85% or more, more preferably 90%or more, 95% or more, or 98% or more of the wild-type polypeptide (amature form in the case of a secretory protein).

Variants of amino acid sequences can be prepared, for example, byintroducing mutations to the DNAs encoding the natural polypeptide(Walker and Gaastra, eds. Techniques in Molecular Biology (MacMillanPublishing Company, New York, 1983); Kunkel, Proc. Natl. Acad. Sci. USA82:488-492, 1985; Kunkel et al., Methods Enzymol. 154:367-382, 1987;Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Plainview, N.Y.), 1989; U.S. Pat. No.4,873,192). An example of guidance for substituting amino acids withoutaffecting biological activity includes the report by Dayhoff et al.(Dayhoff et al., in Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), 1978).

The number of amino acids that are modified is not particularly limited,but for example, it is 30% or less, preferably 25% or less, morepreferably 20% or less, more preferably 15% or less, more preferably 10%or less, 5% or less, or 3% or less of all amino acids of thenaturally-derived mature polypeptide, and is, for example, 15 aminoacids or less, preferably ten amino acids or less, more preferably eightamino acids or less, more preferably five or less, or more preferablythree amino acids or less. When substituting amino acids, activities ofthe protein can be expected to be maintained by substitution to an aminoacid with similar side chain properties. Such substitutions are calledconservative substitutions in the present invention. Examples ofconservative substitutions include substitution and such among aminoacids within each of the groups such as basic amino acids (such aslysine, arginine, and histidine), acidic amino acids (for example,aspartic acid and glutamic acid), uncharged polar amino acids (forexample, glycine, asparagine, glutamine, serine, threonine, tyrosine,and cysteine), nonpolar amino acids (for example, alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, andtryptophan), β-branched amino acids (for example, threonine, valine,isoleucine), and aromatic amino acids (for example, tyrosine,phenylalanine, tryptophan, and histidine). Furthermore, examples includesubstitution among amino acids whose relationship in the BLOSUM62substitution matrix (S. Henikoff and J. G. Henikoff, Proc. Acad. Natl.Sci. USA 89: 10915-10919, 1992) is positive.

The modified proteins exhibit a high homology to the amino acid sequenceof the wild-type protein. High homology refers to amino acid sequenceshaving, for example, 70% or higher, 75% or higher, 80% or higher, 85% orhigher, 90% or higher, 93% or higher, 95% or higher, or 96% or higheridentity. Amino acid sequence identity can be determined using, forexample, the BLASTP program (Altschul, S. F. et at, J. Mol. Biol.215:403-410, 1990). A search can be carried out using default parametersin the Web page of BLAST at NCBI (National Center for BiotechnologyInformation) (Altschul S. F. et al., Nature Genet. 3:266-272, 1993;Madden, T. L. et al., Meth. Enzymol. 266:131-141, 1996; Altschul S. F.et al., Nucleic Acids Res. 25:3389-3402, 1997; Zhang J. & Madden T. L.,Genome Res. 7:649-656, 1997). Alignment of two sequences can beproduced, for example, by the Blast 2 sequences program which comparestwo sequences (Tatiana A et al., FEMS Microbiol Lett. 174:247-250, 1999)and the identity of the sequences determined. Gaps and mismatches aretreated similarly, and for example, a value of identity with respect tothe entire amino acid sequence of a naturally-derived cytokine (matureform after secretion) is calculated. Specifically, the proportion of thenumber of matching amino acids in the total number of amino acids of thewild-type protein (mature form in the case of a secreted protein) iscalculated.

Furthermore, genes can be introduced with a silent mutation such thatthe encoded amino acid sequence is not changed. Particularly, in AT richgenes, by substituting five or more consecutive A or T nucleotides withG or C such that the encoded amino acid sequence is not changed, highexpression of genes can be stably obtained.

Examples of modified proteins or proteins used for reprogramming areproteins encoded by nucleic acids that hybridize under stringentconditions with a part or all of the coding region of a gene encodingthe wild-type protein and having an activity (activity to inducereprogramming) equivalent to that of the wild-type protein. Inhybridization, for example, a probe is prepared either from a nucleicacid comprising a sequence of the coding region of the wild-type proteingene or a complementary sequence thereof or from a nucleic acid which isthe object of hybridization, and identification can be carried out bydetecting whether or not the probe hybridizes to the other nucleic acid.Stringent hybridization conditions are, for example, conditions ofperforming hybridization in a solution containing 5×SSC, 7% (WN) SDS,100 micro-g/mL denatured salmon sperm DNA, 5×Denhardt's solution(1×Denhardt's solution includes 0.2% polyvinyl pyrrolidone, 0.2% bovineserum albumin, and 0.2% Ficoll) at 60° C., preferably 65° C., and morepreferably 68° C., and then washing by shaking for two hours in 2×SSC,preferably in 1×SSC, more preferably in 0.5×SSC, and more preferably in0.1×SSC at the same temperature as hybridization.

Examples of genes particularly preferable for inducing cellularreprogramming include F-box protein 15 (Fbx15, NM_152676, NM_015798),Nanog (NM_024865, AB093574), ERAS (ES cell expressed Ras; NM_181532,NM_181548), DPPA2 (NM_138815, NM_028615), Oct3/4 (also called POU5F1;NM_002701, NM_203289, NM_013633, NM_001009178), Sox2 (NM_003106,NM_011443, XM_574919), TCL1A (T-cell leukemia/lymphoma 1A; NM_021966,NM_009337), KLF4 (Kruppel-like factor 4; NM_004235, NM_010637), cateninβ1(cadherin-associated protein beta 1; NM_001904, NM_007614; includingthe S33Y mutant), and c-Myc (NM_002467, NM_010849; including the T58Amutant), as well as other genes which are members of the families towhich these genes belong. When these genes are introduced, theproportion of colonies showing the morphology of induced pluripotentstem cells has been reported to be higher than when the four types ofgenes (Oct3/4, Sox2, KLF4, and c-Myc) described next are introduced(WO2007/69666). Therefore, chromosomally non-integrating virus vectorscarrying any one of these are useful for use in introducing cellularreprogramming in the present invention, and in particular, they can beused favorably for inducing pluripotent stem cells. Individual virusvectors can be used by combining them at the time of use. Furthermore,they can be combined in advance to form a kit, or they may be mixed toform a composition. Furthermore, one or more chromosomallynon-integrating virus vectors containing any combination (or all) ofthese genes, and kits or compositions containing these vectors are alsoincluded in the present invention.

Among them, a combination of genes particularly preferred for inductionof pluripotent stem cells is a combination comprising at least fourtypes of genes which are the Sox gene, the KLF gene, the Myc gene, andthe Oct gene (Takahashi, K. and Yamanaka S., Cell 126, 663-676, 2006;Lowry W E et al., Proc Natl Acad Sci USA, 105(8):2883-8, 2008; Masaki,H. et al., Stem Cell Res. 1:105-115, 2008; WO2007/69666). Herein, theSox protein, the KLF protein, the Myc protein, and the Oct protein, andtheir genes refer to proteins and genes which are members belonging tothe Sox family, the KLF family, the Myc family, and the Oct family,respectively. There are reports that by making adjustments so that oneor more members from each of these four families are expressed,pluripotent stem cells can be induced from various differentiated cells.For example, regarding the Sox family genes, the use of any of the Sox1,Sox2, Sox3, Sox15, and Sox17 genes has been reported to be able toinduce pluripotent stem cells (WO2007/69666). Regarding the KLF familyas well, pluripotent stem cells could be induced with KLF4 or KLF2(WO2007/69666). Regarding the Myc family as well, not only the wild-typec-Myc but the T58A mutant, N-Myc, and L-Myc could also inducepluripotent stem cells (WO2007/69666; Blelloch R. et al., Cell StemCell, 1: 245-247, 2007). This way, since genes of the families can beselected in various ways and then used, reprogramming can be induced byappropriately selecting genes from the four families mentioned above.

For example, the amount of expression of wild-type c-Myc from RNA virusvectors such as Sendai virus vectors was found to be low. However, byintroducing one or more, preferably two or more, three or more, four ormore, or all five mutations selected from among a378g, t1122c, t1125c,a1191g, and a1194g into wild-type cMyc, the gene can be highly expressedwith stability from the vector. In the present invention, for example, amodified c-Myc gene indicated in SEQ ID NO: 45 can be used favorably.The position where the gene is inserted in the vector can be can beselected as desired.

For example, the Myc gene may be positioned at the rear (5′ side) of theminus-strand RNA genome, that is, at a position that can be locatedfaster from the 5′ side than from the 3′ side among the multipleprotein-encoding sequences positioned on the genome (see the Examples).The Myc gene can be positioned, for example, closest to the 5′ side(that is, at the first position from the 5′ side), or at the second orthird position from the 5′ side. The Myc gene can be positioned, forexample, at the second position from the 5′ side of the genome, orspecifically, when the L gene is positioned closest to the 5′ side ofthe genome and the HN gene is positioned next, the Myc gene can bepositioned between them. The Myc gene can have substitutions in thecontinuous A or T nucleotide sequence by introduction of suitable silentmutations such that the encoded amino acid sequence is not changed.

A minus-strand RNA virus vector having the Myc gene positioned at therear (5′ side) of the minus-strand RNA genome can be used in combinationwith other nuclear reprogramming factor-encoding minus-strand RNA virusvectors. In this case, in the other nuclear reprogrammingfactor-encoding minus-strand RNA virus vectors, the nuclearreprogramming factors can be positioned at the front (3′ side) of theminus-strand RNA genome of the respective vectors, that is, at aposition that can be located faster from the 3′ side than from the 5′side among the multiple protein-encoding sequences positioned on thegenome. For example, they may be positioned closest to the 3′ side (thatis, at the first position from the 3′ side), or at the second or thirdposition from the 3′ side. For example, genes encoding nuclearreprogramming factors other than Myc (for example, the Oct gene, Klfgene, and Sox gene) are positioned at first or second, or morepreferably at the first position from the 5′ side of the genome in therespective minus-strand RNA virus vectors. Specifically, a gene encodingthe nuclear reprogramming factor can be positioned at the most 3′ endside on the 3′ side of the NP gene of the genome.

From the colonies of cells which have completed reprogramming, cellsfrom which the vectors have been removed can be selected appropriately.For example, cells from which the vectors have been naturally removedmay be selected. To this end, for example, negative selection can becarried out using antibodies specific to the virus vectors (for example,anti-HN antibodies). Furthermore, when using temperature-sensitivevectors, the vectors can be removed easily by culturing at hightemperatures (for example, 37.5° C. to 39° C., preferably 38° C. to 39°C., or 38.5° C. to 39° C.).

Specifically, the KLF family includes Klf1 (NM_006563, NM_010635), Klf2(NM_016270, NM_008452), Klf4 (NM_004235, NM_010637), and Klf5(NM_001730, NM_009769); the Myc family includes c-Myc (NM_002467,NM_010849, including the T58A mutant), N-Myc (NM_005378, NM_008709), andL-Myc (NM_005376, NM_005806); the Oct family includes Oct1A (NM_002697,NM_198934), Oct3/4 (NM_002701, NM_203289, NM_013633, NM_001009178), andOct6 (NM_002699, NM_011141); and the Sox family includes Sox1(NM_005986, NM_009233), Sox2 (NM_003106, NM_011443, XM_574919), Sox3(NM_005634, NM_009237), Sox1 (NM_031439, NM_011446), Sox15 (NM_006942,NM_009235), Sox17 (NM_022454, NM_011441), and Sox18 (NM_018419,NM_009236). Chromosomally non-integrating virus vectors carrying any oneof these genes are useful for use in inducing dedifferentiation of cellsin the present invention, and can be used favorably for induction ofpluripotent stem cells in particular.

Myc family genes are not essential for induction of pluripotent stemcells, and pluripotent stem cells can be induced using only the genes ofthe three families excluding the Myc family genes (Nakagawa M. et al.,Nat Biotechnol. 26(1):101-6, 2008; Wering M. et al., Cell Stem Cell2(1):10-2, 2008; Example 5). When the Myc gene is not expressed, forexample, p53 siRNA and UTF1 can be used to significantly increase theinduction efficiency of pluripotent stem cells (Y. Zhao et al., CellStem Cell, 3 (5): 475-479, 2008; N. Maherali, and K. Hochedlinger, CellStem Cell, 3 (6): 595-605, 2008). Furthermore, induction of pluripotentstem cells has been also reported to be possible using only the genes ofthe three families excluding the KLF family genes (Park I H et al.,Nature, 451(7175):141-6, 2008). In addition, by combined use of the G9ahistone methyltransferase inhibitor (BIX-01294; Kubicek, S. et al., Mol.Cell 25, 473-481, 2007), induction of pluripotent stem cells has beenreported to be possible from fetal NPC using only three genes, i.e., theKlf gene, the Sox gene, and the Myc gene (Shi Y et al., Cell Stem Cell,2(6):525-8, 2008). Therefore, one or a number of chromosomallynon-integrating virus vectors carrying any of the Sox gene, the KLFgene, and the Oct gene, or any of the Sox gene, the Myc gene, and theOct gene, or a combination of the Sox gene, the Myc gene, and the Klfgene are specially useful for use in inducing cellular reprogramming inthe present invention, and can be used favorably for inducingpluripotent stem cells. Virus vectors that encode the respective genescan be separately prepared individually. They can be used by combiningthem at the time of use. Any combination or all of them may be combinedto form a kit or mixed to form a composition. Furthermore, the presentinvention relates to one or more chromosomally non-integrating virusvectors comprising any combination (or all) of these genes, and a kit ora composition for reprogramming which comprise these vectors.Furthermore, a portion of the recombinant vectors included in this kitcan be substituted with proteins, synthetic compounds, or such havingcorresponding functions.

When one or several of the above-mentioned genes are, for example,already expressed endogenously in the original differentiated cells,introduction of those genes can be omitted. For example, since neuralprogenitor cells (NPCs) express endogenous Sox family genes, pluripotentstem cells can be induced by the introduction of only Oct3/4 and Klf4(Shi Y et al., Cell Stem Cell, 2(6):525-8, 2008). Furthermore, inductionof pluripotent stem cells from mouse embryonic fibroblasts (MEF) usingthree genes, Oct4, Sox2, and Esrrb (estrogen-related receptor beta,NM_004452.2, NP 004443.2, NM_011934.3, NP 036064.2) has been reported tobe possible, and it has been suggested that Esrrb is able to complementthe function of Klf (Feng, B. et al., Nat Cell Biol. 11(2):197-203,2009). Furthermore, by combining a histone methyltransferase inhibitor(BIX-01294) and a calcium ion channel agonist (BayK8644), pluripotentstem cells can be induced from embryonic fibroblasts by the introductionof only Oct3/4 and Klf4 (Shi Y et al., Cell Stem Cell, 3(5):568-574,2008). In experiments using neural stem cells (NSCs) derived from adultmouse brain, the introduction of not only the combination of Oct3/4 andKlf4, but also of only the genes of two factors, Oct3/4 and c-Myc, hasbeen reported to be able to induce pluripotent stem cells (Kim, J. B. etal., Nature, doi: 10.1038/nature07061; Published online 29 Jun. 2008;Nature. 2008, 454(7204):646-50). Furthermore, by adjusting the culturingperiod, pluripotent stem cells can be induced using Oct4 alone (JeongBeom Kim et al., Cell, 136(3): 411-419, 2009). As for chromosomallynon-integrating virus vectors encoding reprogramming factors, only thosenecessary can be appropriately used. Furthermore, if endogenousexpression of endogenous reprogramming factors is induced by theexpression of other genes, by chemical treatment, or such, introductionof a vector expressing such other genes or chemical treatment may becombined with the introduction of only the chromosomally non-integratingvirus vectors encoding reprogramming factors that cannot be induced byjust the above treatments. In the present invention, combining vectorsso that at least the three types of genes of the Oct gene, the Klf gene,and the Sox gene, at least the four types of genes of the Oct gene, theKlf gene, the Sox gene, and the Myc gene, or at least the four types ofgenes of the Oct gene, the Sox gene, the Nanog gene, and the Lin28 geneare expressed endogenously or exogenously includes, for example, notonly states in which certain reprogramming factors are endogenouslyexpressed in a natural state, but also includes cases where, in the casethe expression of endogenous reprogramming factors can be induced byintroduction of vectors expressing other genes or by chemical treatment,protein treatment, or such, combinations of these treatments arecombined with chromosomally non-integrating virus vectors so that justthe lacking factors are exogenously expressed.

Furthermore, besides the combinations of the four types or three typesmentioned above, combinations which include each of the four types ofgenes of the Oct gene, the Sox gene, the NANOG gene (NM_024865,AB093574) and the L1N28 gene (NM_024674) are also useful for inductionof pluripotent stem cells (Yu J. et al., Science, 318(5858):1917-20,2007). Combinations produced by further combining the Myc gene and theKLF gene are also favorable (Liao J et al., Cell Res. 18(5):600-3,2008). Chromosomally non-integrating virus vectors carrying any one ofthese genes are particularly useful in the present invention for use inthe induction of cellular dedifferentiation, and can be used favorablyfor the induction of pluripotent stem cells. One or more chromosomallynon-integrating virus vectors containing any combination (or all) ofthese genes, and kits or compositions comprising these vectors can alsobe used favorably in cellular reprogramming, and particularly in theproduction of pluripotent stem cells. Meanwhile, similarly as describedabove, when the subject cells already express a portion of these genes,vectors expressing those genes do not have to be introduced.Furthermore, a portion of the recombinant vectors included in this kitmay be substituted with proteins, synthetic compounds, and such thathave corresponding functions.

Other genes can be further combined to the above-described combinationof genes to increase the efficiency of induction of reprogramming.Examples of such genes include TERT (NM_198253, NM_009354) and/or SV40large T antigen (NC_001669.1, Fiers, W. (05-11-1978) Nature 273: (5658)113-120) (Park I H. et al., Nature, 451 (7175):141-6, 2008). One or moregenes selected from the group consisting of HPV16 E6, HPV16 E7, and Bmil(NM_005180, NM_007552) may also be further combined. Furthermore, one orany combination of genes selected from the group consisting of Fbx15(Mol Cell Biol. 23(8):2699-708, 2003), Nanog (Cell 113: 631-642, 2003),ERas (Nature 423, 541-545, 2003), DPPA2 (Development 130: 1673-1680,2003), TCL1A (Development 130: 1673-1680, 2003), and β-Catenin (Nat Med10(1): 55-63, 2004) may be expressed. In addition, one or more genesselected from the group consisting of ECAT1 (AB211062, AB211060), DPPA5(NM_001025290, NM_025274, XM_236761), DNMT3L (NM_013369, NM_019448),ECAT8 (AB211063, AB211061), GDF3 (NM_020634, NM_008108), SOX15(NM_006942, NM_009235), DPPA4 (NM_018189, NM_028610), FTHL17 (NM_031894,NM_031261), SALL4 (NM_020436, NM_175303), Rex-1 (NM_174900, NM_009556),Utf1 (NM_003577, NM_009482), DPPA3 (NM_199286, NM_139218, XM_216263),STAT3 (NM_139276, NM_213659), and GRB2 (NM_002086, NM_008163) may becombined. By additionally expressing these genes, induction ofpluripotent stem cells may be promoted (WO2007/69666). When mature Bcells are the subjects, for example, the myelocytic transcription factorC/EBPα (CCAAT/enhancer-binding-protein α) (NM_004364) can be ectopicallyexpressed, or expression of the B cell transcription factor Pax5 (pairedbox 5; NM_016734) can be suppressed to promote reprogramming (Hanna J,Cell. 133(2):250-64, 2008). These factors can also be expressed usingthe chromosomally non-integrating virus vectors of the presentinvention. Furthermore, a portion of the recombinant vectors included inthis kit can be substituted with proteins, synthetic compounds, and suchwhich have corresponding functions.

Furthermore, besides expressing the above-mentioned factors, forexample, by combining the addition of compounds, the efficiency ofreprogramming can be increased. For example, bFGF (basic fibroblastgrowth factor) and/or SCF (stem cell factor) can promote the inductionof pluripotent stem cells, and moreover can replace the function ofc-Myc in the induction of pluripotent stem cells (WO2007/69666).Furthermore, MAP kinase inhibitors (PD98056) are also useful forestablishing pluripotent stem cells that are closer to ES cells, andsuch (WO2007/69666). Furthermore, DNA methylase (Dnmt) inhibitors and/orhistone deacetylase (HDAC) inhibitors are reported to improve theefficiency of induction of pluripotent stem cells (Huangfu D et al., NatBiotechnol. (Published online: 22 Jun. 2008, doi:10.1038/nbt1418); Nat.Biotechnol. 26, 795-797 (2008)). For example, combined use of HDAC(VPA)enables induction of pluripotent stem cells by introduction of only twogenes, Oct4 and Sox2 (Huangfu, D. et al., Nat Biotechnol. 200826(11):1269-75). Vectors of the present invention are useful as agentsfor expressing these genes or a portion of those genes. As Dnmtinhibitors, for example, 5-azacytidine and such are useful, and as HDACinhibitors, for example, suberoylanilide hydroxamic acid (SAHA),trichostatin A (TSA), valproic acid (VPA) and such are useful.Furthermore, when using 5-azacytidine, combined use of glucocorticoid(dexamethasone) can increase the efficiency.

To reprogram cells, the above-mentioned combinations of vectors and suchare introduced into cells. When a number of vectors and/or compounds arecombined and introduced, the introduction is preferably carried outsimultaneously, and specifically, it is preferable to complete theaddition of all vectors encoding the reprogramming factors and/orcompounds within 48 hours or less, preferably 36 hours or less, morepreferably 24 hours or less, 18 hours or less, twelve hours or less, tenhours or less, eight hours or less, six hours or less, three hours orless, two hours or less, or one hour or less from the addition of thefirst vector, compound, or such. The dose of the vectors can be preparedappropriately, but infection is carried out preferably at MOI of 0.3 to100, more preferably at MOI of 0.5 to 50, more preferably at MOI of 1 to30, more preferably at MOI of 1 to 10, more preferably at MOI of 1 to 5,and more preferably at MOI of approximately 3. The induced pluripotentstem cells form flat colonies very similar to those of ES cells, andexpress alkaline phosphatase. Furthermore, the induced pluripotent stemcells may express the undifferentiated-cell markers Nanog, Oct4, and/orSox2, and the like. The induced pluripotent stem cells preferably showTERT expression and/or telomerase activity. The present invention alsorelates to methods for producing cells that express alkaline phosphataseand preferably further express Nanog and/or TERT which areundifferentiated-cell markers, and to a use of chromosomallynon-integrating virus vectors in the production of these cells and inthe production of pharmaceutical agents for inducing these cells.

According to the present invention, colonies of pluripotent stem cellscan be induced from desired cells including adult skin cells andneonatal foreskin cells, for example at an incidence rate of 0.3×10⁻⁵ ormore, 0.5×10⁻⁵ or more, 0.8×10⁻⁵ or more, or 1×10⁻⁵ or more (forexample, 1.7×10⁻⁵ to 2.4×10⁻³), and preferably at an incidence rate of1.5×10⁻⁵ or more, 1.7×10⁻⁵ or more, 2.0×10⁻⁵ or more, 2.5×10⁻⁵ or more,3×10⁻⁵ or more, 4×10⁻⁵ or more, 5×10⁻⁵ or more, 8×10⁻⁵ or more, 1×10⁻⁴or more, 2×10⁻⁴ or more, 3×10⁻⁴ or more, 5×10⁻⁴ or more, 8×10⁻⁴ or more,1×10⁻³ or more, 1.5×10⁻³ or more, 2×10⁻³ or more, or 2.3×10⁻³ or more.

Differentiated cells which become the object of induction ofreprogramming are not particularly limited, and desired somatic cellsand such may be used. Production of pluripotent stem cells from somaticcells has been shown to be possible not only from cells derived fromfetal mice but also from differentiated cells collected from the tailportion of adult mice, and from liver cells, and gastric mucosal cells,and this suggests that the production is not dependent on the cell typeor the state of differentiation (WO2007/069666; Aoi T. et al., Science[Published Online Feb. 14, 2008]; Science. 2008; 321(5889):699-702).Induction of pluripotent stem cells has been confirmed to be possible inhumans as well, from various cells such as adult facial skin-derivedfibroblasts, adult synoviocytes, neonatal foreskin-derived fibroblasts,adult mesenchymal stem cells, skin cells from the palm of an adult, andembryonic cells (Takahashi K et al. (2007) Cell 131: 861-872; Park I Het al., Nature, 451(7175):141-6, 2008). Furthermore, induction ofpluripotent stem cells has been reported similarly from terminallydifferentiated cells such as pancreatic β cells and B lymphocytes aswell (Stadtfeld M et al., Curr Biol. 2008 May 21. [PubMed, PMID:18501604]; Curr Biol. 2008; 18(12):890-4; Hanna J. et at, Cell.133(2):250-64, 2008). These findings suggest that induction ofpluripotent stem cells do not depend on the cells serving as the origin.Methods of the present invention can be applied in the induction ofpluripotent stem cells from these desired somatic cells. Specifically,differentiated cells which are the object of reprogramming includefibroblasts, synoviocytes, mucosal cells of the oral cavity, stomach, orsuch, liver cells, bone marrow cells, tooth germ cells, and otherdesired cells. Furthermore, cells may be derived, for example, fromcells of embryos, fetuses, newborns, children, adults, or the aged. Theorigin of the animals is not particularly limited, and includes mammalssuch as humans and non-human primates (monkeys and such), rodents suchas mice and rats, and non-rodents such as bovine, pigs, and goats.

Cells produced by the methods of the present invention are useful forcausing differentiation into a variety of tissues and cells, and can beused in desired examinations, research, diagnosis, tests, treatments,and such. For example, induced stem cells are expected to be utilized instem cell therapy. For example, reprogramming is induced by usingsomatic cells collected from patients, and then somatic stem cells andother somatic cells that are obtained by induction of differentiationcan be transplanted into patients. Methods for inducing cellulardifferentiation are not particularly limited, and for example,differentiation can be induced by retinoic acid treatment, treatmentwith a variety of growth factors/cytokines, and treatment with hormones.Furthermore, the obtained cells can be used for detecting effects of thedesired pharmaceutical agents and compounds, and this enables screeningof pharmaceutical agents and compounds to be carried out.

EXAMPLES

Hereinbelow, the present invention is specifically described withreference to the Examples; however, it is not to be construed as beinglimited thereto. All documents and other references cited herein areincorporated as part of this description.

<Construction of Sendai Virus Vectors Carrying a Foreign Gene Used inthe Present Invention>

The methods for constructing Sendai virus vectors carrying a foreigngene used in the present invention are described below. Unless otherwisespecified, foreign genes were introduced using the vectors. Hereinbelow,“SeV18+/TSΔF” refers to an F gene-deficient Sendai virus vector in whichthe M protein has the G69E, T116A, and A183S mutations; the FIN proteinhas the A262T, G264, and K461G mutations; the P protein has the L511Fmutation; and the L protein has the N1197S and K1795E mutations(WO2003/025570). This vector has an insertion site (NotI site) for anintroduced gene upstream of the NP gene (on the 3′ side of the genome;also referred to as “position 18+”).

(1) Construction of cDNA Libraries for Isolation of the c-Myc, Sox2,KLF4, and Oct3/4 Genes

Total RNA was extracted from Jurkat cells to isolate the c-Myc, Sox2,KLF4, and Oct3/4 genes. 1.0×10⁶ Jurkat cells (Schneider U et al. (1977)Int J Cancer 19(5):621-6) were collected by centrifugation at 8,000 rpmand room temperature for one minute. 200 μl of a cell lysis buffer (10mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl₂, 0.65% NP-40) was addedto the cells. After pipetting, the cells were suspended by vortexing.Following centrifugation at 6,000 rpm for three minutes, the supernatantwas transferred to another 1.5-ml Eppendorf tube. 200 μl of anextraction buffer was add thereto, and this was sufficiently suspendedby vortexing. Then, 400 μl of phenol/chloroform/isoamyl alcohol(25:24:1) was added thereto, and this was sufficiently suspended byvortexing. After centrifugation at 15,000 rpm and 4° C. for fiveminutes, the supernatant was transferred to another 1.5-ml Eppendorftube. Then, 400 μl of isopropanol was added thereto, and this wassufficiently suspended by vortexing. This suspension was cooled at −20°C. for 30 minutes. Following centrifugation at 15,000 rpm and 4° C. for15 minutes, the supernatant was discarded, and 1 ml of 70% ethanol wasadded to the precipitate. After suspension by vortexing, this wascentrifuged at 15,000 rpm and 4° C. for five minutes. The supernatantwas discarded, and the precipitate was dried at room temperature. Then,this was dissolved in 100 μl of nuclease-free water to prepare a Jurkatcell total RNA solution.

To isolate the KLF4 gene, total RNA was prepared from 293T/17 cellsderived from human embryonic kidney cells (Human embryonic kidneysubclone 17; ATCC CRL-11286; Pear, W. S. et al., 1993, Proc. Natl. Acad.Sci. USA 90:8392-8396) by the same method as described above.

To isolate the Oct3/4 gene, total RNA was prepared from NCCIT cellswhich are human embryonic carcinoma cells (ATCC number CRL-2073;Damjanov I, et al., Lab. Invest. 1993, 68(2): 220-32) by the same methodas described above.

Using SuperScript III Reverse Transcriptase (Invitrogen, catalog No.18080-044), cDNAs were synthesized from the prepared total RNAs. 1 μg oftotal RNA was mixed with 100 ng of random hexamer and 1 μl of 10 mM dNTPmixture, and the total volume was adjusted to 13 μl with nuclease-freewater. After heat treatment at 65° C. for five minutes, the mixture wascooled on ice for one minute. Then, 4 μl of 5× First-Strand Buffer, 1 μlof 0.1 M DTT, 1 μl of RNaseOUT, and 1 μl of Superscript III RT wereadded thereto. After mixing by pipetting, the mixture was spinned down.Then, this was incubated at 25° C. for five minutes at 50° C. for 60minutes, and then at 70° C. for 15 minutes. 180 μl of TE (pH8.0) wasadded thereto, and this was used as a cDNA library.

(2) Isolation of the Human Transcriptional Factor c-Myc, andConstruction of a Sendai Virus Vector Plasmid Carrying c-Myc

The Jurkat cDNA library was subjected to PCR (94° C. for three minutes,and 40 cycles of [98° C. for 10 seconds, 55° C. for 15 seconds, and 72°C. for two minutes], followed by 72° C. for seven minutes) usingPrimeStar™ HS DNA polymerase (Takara Bio, catalog No. R010A) and thefollowing primers: c-Myc-21F (5′-AACCAGCAGCCTCCCGCGACG-3′ (SEQ ID NO:1)) and c-Myc 1930R (5′-AGGACATTTCTGTTAGAAGGAATCG-3′ (SEQ ID NO: 2)).The PCR product was diluted 100-fold with TE, and a 1-μl aliquot wassubjected to PCR using the following primers: c-Myc-F(5′-GATGCCCCTCAACGTTAGCTTCACC-3′ (SEQ ID NO: 3)) and c-Myc-R(5′-GTTACGCACAAGAGTTCCGTAGCTG-3′ (SEQ ID NO: 4)). The PCR product wasseparated by electrophoresis using 1% agarose gel. A band of about 1.3kbp was excised, and the DNA was purified using a Qiaquick GelExtraction Kit (QIAGEN, Cat. No. 28706). This was cloned into the SwaIsite of pCAGGS-BSX (WO2005/071092). A clone that has the correctsequence was selected by sequencing, and thus pCAGGS-BSX-c-Myc wasobtained. Then, PCR was carried out using pCAGGS-BSX-c-Myc as atemplate, together with the following primers: NotI-c-Myc F(5′-ATTGCGGCCGCATGCCCCTCAACGTTAGCTTCAC-3′ (SEQ ID NO: 5)) and NotI-c-MycR (5′-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTACGCACAAGAGTTCCGTAGCTGTTCAAGTTTGTGTTTC-3′ (SEQ ID NO: 6)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and then this was digested with NotI at 37° C. for three hours.The digest was purified using a Qiaquick PCR Purification kit (QIAGEN,catalog No. 28106), and this was cloned into the NotI site of aBluescript plasmid vector. The gene sequence was determined bysequencing. A clone that has the correct sequence was selected, and thuspBS-KS-c-Myc was obtained. pBS-KS-c-Myc was digested with NotI at 37° C.for three hours, and this was separated by electrophoresis using 1%agarose gel. A band of about 1.5 kbp was excised, and the DNA waspurified using a Qiaquick Gel Extraction Kit (QIAGEN, catalog No.28706). The NotI fragment containing the c-Myc gene was cloned into theNotI site of the pSeV18+/TSΔF vector encoding the antigenome of a Sendaivirus vector (SeV18+/TSΔF). A clone that has the correct sequence wasselected by sequencing, and thus pSeV18c-Myc/TSΔF was obtained.

(3) Isolation of the Human Transcriptional Factor SOX2 Gene andConstruction of a Sendai Virus Vector Plasmid Carrying the SOX2 Gene

The Jurkat cDNA library was subjected to PCR (94° C. for three minutes,and 40 cycles of [98° C. for 10 seconds, 55° C. for 15 seconds, and 72°C. for two minutes], followed by 72° C. for seven minutes) usingPrimeStar HS DNA polymerase (Takara Bio, catalog No. R010A) and thefollowing primers: SOX2-64F (5′-CAAAGTCCCGGCCGGGCCGAGGGTCGG-3′ (SEQ IDNO: 7)) and SOX2-1404R (5′-CCCTCCAGTTCGCTGTCCGGCCC-3′ (SEQ ID NO: 8)).The PCR product was diluted 100-fold with TE, and a 1-μl aliquot wassubjected to PCR using the following primers: Sox2-F(5′-GATGTACAACATGATGGAGACGGAGC-3′ (SEQ ID NO: 9)) and Sox2-R(5′-GTCACATGTGTGAGAGGGGCAGTG-3′ (SEQ ID NO: 10)). The PCR product wasseparated by electrophoresis using 1% agarose gel. A band of about 0.95kbp was excised, and the DNA was purified using a Qiaquick GelExtraction Kit (QIAGEN, Cat. No. 28706). This was cloned into the SwaIsite of pCAGGS-BSX. A clone that has the correct sequence was selectedby sequencing, and thus pCAGGS-BSX-SOX2 was obtained. Then, PCR wascarried out using pCAGGS-BSX-SOX2 as a template, together with thefollowing primers: Not I Sox-2F (5′-ATTGCGGCCGCATGTACAACATGATGGAGACG-3′(SEQ ID NO: 11)) and Not I Sox-2R(5′-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCACATGTGTGAGAGGGGCAGTGTGCCGTTAATGGCCGTG-3′ (SEQ ID NO: 12)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and then digested with NotI at 37° C. for three hours. Thedigest was purified using a Qiaquick PCR Purification kit (QIAGEN,catalog No. 28106), and this was cloned into the NotI site of aBluescript plasmid vector. The gene sequence was determined bysequencing. A clone that has the correct sequence was selected, and thuspBS-KS-Sox2 was obtained. pBS-KS-Sox2 was digested with NotI at 37° C.for three hours, and this was separated by electrophoresis using 1%agarose gel. A band of about 1 kbp was excised, and the DNA was purifiedusing a Qiaquick Gel Extraction Kit (QIAGEN, catalog No. 28706). TheNotI fragment containing the Sox2 gene was cloned into the NotI site ofthe pSeV18+/TSΔF vector. A clone that has the correct sequence wasselected by sequencing, and thus pSeV18+Sox2/TSΔF was obtained.

(4) Isolation of the Human Transcriptional Factor KLF4 Gene andConstruction of a Sendai Virus Vector Plasmid Carrying the KLF4 Gene

The Jurkat cDNA library was subjected to PCR (94° C. for three minutes,and 40 cycles of [98° C. for 10 seconds, 55° C. for 15 seconds, and 72°C. for two minutes], followed by 72° C. for seven minutes) usingPrimeStar HS DNA polymerase (Takara Bio, catalog No. R010A) and thefollowing primers: KIF-4-35F (5′-CCACATTAATGAGGCAGCCACCTGGC-3′ (SEQ IDNO: 13)) and KIF-4 1772R (5′-GCAGTGTGGGTCATATCCACTGTCTG-3′ (SEQ ID NO:14)). The PCR product was diluted 100-fold with TE, and a 1-μl aliquotwas subjected to PCR using the following primers: KIF4-F(5′-GATGGCTGTCAGCGACGCGCTGCTCCC-3′ (SEQ ID NO: 15)) and KIF4-R(5′-GTTAAAAATGCCTCTTCATGTGTAAGGCGAG-3′ (SEQ ID NO: 16)). The PCR productwas separated by electrophoresis using 1% agarose gel. A band of about1.4 kbp was excised, and the DNA was purified using a Qiaquick GelExtraction Kit (QIAGEN, Cat. No. 28706). This was cloned into the SwaIsite of pCAGGS-BSX to obtain pCAGGS-BSX-KLF4 #19. The result ofsequencing showed that pCAGGS-BSX-KLF4 #19 has a single silent mutation(c19t). Thus, PCR was carried out using pCAGGS-BSX-KLF4 #19 as atemplate, together with the following primers: NotI-KIF4-F(5′-ATTGCGGCCGCGACATGGCTGTCAGCGACGCGCTG-3′ (SEQ ID NO: 17)) andNotI-KIF4-R (5′-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTAAAAATGCCTCTTCATGTGTAAGGCGAGGTGGTC-3′ (SEQ ID NO: 18)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and this was cloned into the SwaI site of pCAGGS-BSX. A clonethat has the correct sequence was selected by sequencing, and thuspCAGGS-BSX-KLF4 was obtained. Then, PCR was carried out usingpCAGGS-BSX-KLF4 as a template, together with the following primers:NotI-KIF4-F (5′-ATTGCGGCCGCGACATGGCTGTCAGCGACGCGCTG-3′ (SEQ ID NO: 17))and NotI-KIF4-R(5′-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTTAAAAATGCCTCTTCATGTGTAAGGCGAGGTGGTC-3′ (SEQ ID NO: 18)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and then this was digested with NotI at 37° C. for three hours.The digest was purified using a Qiaquick PCR Purification kit (QIAGENcatalog No. 28106), and this was cloned into the NotI site of aBluescript plasmid vector. The gene sequence was determined bysequencing. A clone that has the correct sequence was selected, and thuspBS-KS-KLF4 was obtained. pBS-KS-KLF4 was digested with NotI at 37° C.for three hours, and this was separated by electrophoresis using 1%agarose gel. A band of about 1.5 kbp was excised, and the DNA waspurified using a Qiaquick Gel Extraction Kit (QIAGEN, catalog No.28706). The NotI fragment containing the KLF4 gene was cloned into theNotI site of the pSeV18+/TSΔF vector. A clone that has the correctsequence was selected by sequencing, and thus pSeV18+KLF4/TSΔF wasobtained.

(5) Isolation of the Human Transcriptional Factor Oct3/4 Gene andConstruction of a Sendai Virus Vector Plasmid Carrying the Oct3/4 Gene

Two regions of Oct3/4 were separately amplified by PCR. The NCCIT cDNAlibrary was subjected to PCR (94° C. for three minutes, and 35 cycles of[98° C. for 10 seconds, 55° C. for 15 seconds, and 72° C. for oneminute], followed by 72° C. for seven minutes) using PrimeStar HS DNApolymerase (Takara Bio, catalog No. R010A), together with the followingprimers: Oct-3-28F (5′-CACCATGCTTGGGGCGCCTTCCTTCC-3′ (SEQ ID NO: 19))and OCT3/4 R301 (5′-CATCGGAGTTGCTCTCCACCCCGAC-3′ (SEQ ID NO: 20)), orthe following primers: OCT3/4 F192 (5′-CCCGCCGTATGAGTTCTGTGG-3′ (SEQ IDNO: 21)) and NotI-Oct-3/4R-DPN(5′-GCCGCGGCCGCGTTATCAGTTTGAATGCATGGGAGAGCCCAG-3′ (SEQ ID NO: 22)). Thetwo PCR products were purified using a Qiaquick PCR Purification kit(QIAGEN, catalog No. 28106), and eluted with 100 μl of an elution bufferattached to the kit. The eluates were diluted 50-fold with TE. 1 μl eachof the PCR products was combined and subjected to PCR (94° C. for threeminutes, and 35 cycles of [98° C. for 10 seconds, 55° C. for 15 seconds,and 72° C. for 1.5 minutes], followed by 72° C. for seven minutes) usingthe following primers: Not I-Oct-3/4F(5′-GCCGCGGCCGCACCATGGCGGGACACCTGGCTTC-3′ (SEQ ID NO: 23)) and NotI-Oct-3/4R-DPN (5′-GCCGCGGCCGCGTTATCAGTTTGAATGCATGGGAGAGCCCAG-3′ (SEQ IDNO: 22)). The PCR product was purified using a Qiaquick PCR Purificationkit (QIAGEN, catalog No. 28106) and cloned into the SwaI site ofpCAGGS-BSX. A clone that has the correct sequence was selected bysequencing, and thus pCAGGS-BSX-Oct3/4 was obtained. Then, PCR (94° C.for three minutes, and 25 cycles of [98° C. for 10 seconds, 55° C. for15 seconds, and 72° C. for two minutes], followed by 72° C. for sevenminutes) was carried out using pCAGGS-BSX-Oct3/4 as a template, togetherwith the following primers: Not I-Oct-3/4F(5′-GCCGCGGCCGCACCATGGCGGGACACCTGGCTTC-3′ (SEQ ID NO: 23)) and NotI-Oct-3/4R (5%GCCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAGTTTGAATGCATGGGAGAGCCCAGAGTGGTGAC-3′ (SEQ ID NO: 24)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and then this was digested with NotI at 37° C. for two hours.The digest was purified using a Qiaquick PCR Purification kit (QIAGEN,catalog No. 28106). The NotI fragment containing the Oct3/4 gene wascloned into the NotI site of the pSeV18+/TSΔF vector. A clone that hasthe correct sequence was selected by sequencing, and thuspSeV18+Oct3/4/TSΔF was obtained.

(6) Construction of Sendai Virus Vectors Carrying Human TranscriptionalFactors

On the previous day of transfection, 10⁶ 293 T/17 cells were seeded intoeach well of a 6-well plate, and cultured in a CO₂ incubator (5% CO₂) at37° C. Using 15 μl of TransIT-LT1 (Minis), the 293T/17 cells weretransfected with a mixture of: 0.5 μg of pCAGGS-NP, 0.5 μg of pCAGGS-P4C(−), 2 μg of pCAGGS-L (TDK), 0.5 μg of pCAGGS-T7, 0.5 μg of pCAGGS-F5R(WO2005/071085), and 5.0 μg of an above-described Sendai virus vectorplasmid carrying a human transcriptional factor (pSeV18+c-Myc/TSΔF,pSeV18+Sox2/TSΔF, pSeV18+KLF4/TSΔF, or pSeV18+Oct3/4/TSΔF). The cellswere cultured in a CO₂ incubator at 37° C. for two days. Then, 10⁶ cellsof LLC-MK2/F/A which express the fusion protein (F protein) of Sendaivirus (Li, H.-O. et al., J. Virology 74. 6564-6569 (2000); WO00/70070)were overlaid onto the transfected 293T/17 cells in each well. Then, thecells were cultured in a CO₂ incubator at 37° C. for one day. On thefollowing day, the cell culture medium was removed, and the cells werewashed once with 1 ml of MEM supplemented with penicillin-streptomycin(hereinafter abbreviated as PS/MEM). 1 ml of PS/MEM containing 2.5 μg/mltrypsin (hereinafter abbreviated as Try/PS/MEM) was added to each well.The cells were cultured in a CO₂ incubator at 32° C. for two days. Thecells were continuously cultured while exchanging the medium every threeto four days, and in some cases, passaging with LLC-MK2/F/A cells. Analiquot of the culture supernatant was assessed for vector collection byhemagglutination assay. The culture supernatant was harvested aftersufficient hemagglutination was observed. RNA was extracted from theharvested culture supernatant using a QIAamp Viral RNA Mini Kit (QIAGEN,catalog No. 52906), and then subjected to RT-PCR that targets a regionof the inserted transcription factor. Whether the obtained RT-PCRproduct has the correct nucleotide sequence was confirmed by sequencing.Thus, the following vectors were constructed:

(a) F gene-deficient Sendai virus vector carrying the Oct3/4 gene(hereinafter referred to as “SeV18+Oct3/4/TSΔF vector”)

(b) F gene-deficient Sendai virus vector carrying the Sox2 gene(hereinafter referred to as “SeV18+Sox2/TSΔF vector”)

(c) F gene-deficient Sendai virus vector carrying the KLF4 gene(hereinafter referred to as “SeV18-F Klf4/TSΔF vector”)

(d) F gene-deficient Sendai virus vector carrying the c-Myc gene(hereinafter referred to as “SeV18+c-Myc/TSΔF vector”)

Example 1 Preparation of ES-Like Cells Using Sendai Virus VectorsCarrying Foreign Genes

First, 8.0×10⁵ cells each of human newborn foreskin-derived fibroblast(BJ) (ATCC (http://www.atcc.org); CRL-2522), human adult skin-derivedfibroblast (IIDF) (Applications, Inc. 106-05A-1388; derived from thecheek of a 36-year-old white female), and human fetal lung cell-derivedfibroblast (MRCS; ATCC CCL-171) were cultured in DMEM (GIBCO-BRL,11995)/10% FBS (GIBCO-BRL) in a CO₂ incubator (0.5% CO₂) at 37° C. forone day (DMEM (GIBCO-BRL, 11995)/10% FBS (GIBCO-BRL)).

After culturing, the vectors of (a) to (d) below were added at an MOI of3 to the cultured cells.

(a) SeV18+Oct3/4/TSΔF vector

(b) SeV18+Sox2/TSΔF vector

(c) SeV18+Klf4/TSΔF vector

(d) SeV18+c-Myc/TSΔF vector

After addition of the above vectors, the medium (DMEM (GIBCO-BRL;11995)/10% FBS (GIBCO-BRL)) was exchanged on the next day.

The cells were cultured in a CO₂ incubator (0.5% CO₂) at 37° C. forseven or eight days. Then, the cells into which the vectors wereintroduced were detached with 0.25% trypsin. 5.0×10⁴ to 1.0×10⁶ cellswere cultured on 5.0×10⁵ mitomycin-treated feeder cells (for example,MEF) prepared in gelatin-coated 10-cm culture dishes. On the followingday, the DMEM/10% FBS medium was exchanged with Primate ES Cell CultureMedium (ReproCell; RCHEMD001) supplemented with 4 ng/ml bFGF, and thecells were cultured in a CO₂ incubator (3% CO₂). The medium wasexchanged every one or two days. The medium may be a feedercell-conditioned medium.

Colonies appeared after several days. Human ES cell-like colonies becamevisible after about 20 days of culture (FIG. 1; derived from BJ).

As seen from the photographs shown in FIG. 1, flat colonies, which weresimilar to those of human ES cells and obviously distinct from those offibroblasts (BJ) before induction, were observed (Jikken Igaku(Experimental Medicine) Vol. 26, No. 5 (suppl.) pp. 35-40, 2008). It waspossible to isolate the colonies and culture them on fresh feeder cells.The cells were able to be detached using an ES cell-detaching solution(mixture of trypsin and collagenase; ReproCell, RCHETP002), passaged,and grown.

The experiments described below were further conducted to test whetherthe cells prepared by the above initialization experiment expressundifferentiated markers characteristic of ES cells.

Example 2 Alkaline Phosphatase Staining of Cells Prepared by the AboveInitialization Experiment

The alkaline phosphatase activity, which is an undifferentiated markerfor ES cells, was visualized by staining with NBCT/BCIP (PIERCE;NBT/BCIP, 1-Step, #34042). Colonies stained blue, which were positivefor alkaline phosphatase, were observed (FIG. 2).

Example 3 Assessment of the Expression Levels of Specific Genes in CellsPrepared by the Above Culture

Multiple alkaline phosphatase-positive colonies (ALP(+)) described abovein Example 2 were mixed, and RNA was extracted from them (“ALP(+)” inFIG. 3(a)). Reverse transcription was carried out using random primers.PCR was performed using respective primers (FIG. 3(a)).

The primer sequences are shown below:

Fw:  (SEQ ID NO: 25) 5′-GATCCTCGGACCTGGCTAAGC-3′ and  Rv:(SEQ ID NO: 26) 5′-GCTCCAGCTTCTCCTTCTCCAGC-3′ for Oct3/4; Fw: (SEQ ID NO: 27) 5′-AGCGCTGCACATGAAGGAGCACC-3′ and  Rv: (SEQ ID NO: 28)5′-ATGCGCTGGTTCACGCCCGCGCCCAGG-3′ for Sox2; Fw:  (SEQ ID NO: 29)5′-GCTGCACACGACTTCCCCCTG-3′ and  Rv: (SEQ ID NO: 30)5′-GGGGATGGAAGCCGGGAGGAAGCGG-3′ for KLF4; Fw:  (SEQ ID NO: 31)5′-TCTCAACGACAGCAGCTCGC-3′ and  Rv: (SEQ ID NO: 32)5′-CAGGAGCCTGCCTCTTTTCCACAGA-3′ for c-myc; Fw:  (SEQ ID NO: 33)5′-TACCTCAGCCTCCAGCAGAT-3′ and  Rv: (SEQ ID NO: 34)5′-TGCGTCACACCATTGCTATT-3′ for Nanog;  and Fw:  (SEQ ID NO: 35)5′-CAACCGCGAGAAGATGAC-3′ and  Rv: (SEQ ID NO: 36)5′-AGGAAGGCTGGAAGAGTG-3′ for β-actin.

Furthermore, single ES-like cell colonies were isolated and RT-PCR wascarried out by the same method as described above (“4BJ-liPS” in FIG.3(b)). At the same time, hTERT expression was also assessed using thefollowing primers:

Fw: 5′-TGCCCGGACCTCCATCAGAGCCAG-3′ (SEQ ID NO: 37) and Rv:

5′-TCAGTCCAGGATGGTCTTGAAGTCTG-3′ (SEQ ID NO: 38).

The c-Myc expression level was elevated, and the expression ofintroduced genes (Oct3/4, Sox2, and Klf4), which was not detectable infibroblasts (BJ) before induction, was detected. It was also revealedthat the expression of Nanog, which is an ES cell marker, was induced(“ALP(±)” in FIG. 3(a)) as in embryonic carcinoma cells (NCCIT) as apositive control. Nanog is a newly identified homeo-domain protein(Cell, Vol. 113, 631-642, 2003), which is specifically expressed inpluripotent stem cells such as ES and EG cells, and early embryos. Nanogis involved in the signal transduction system for the pluripotency andmaintenance of autonomous replication ability. Furthermore, the cellsderived from single colonies were demonstrated to express theundifferentiated ES cell marker genes and hTERT, which is an indicatorfor telomerase activation that shows the ability of infiniteproliferation (“4BJ-1iPS” in FIG. 3(b)). The above findings support thatthe cells isolated from the colonies were pluripotent stem cells.

Example 4 Preparation of Inducible Pluripotent Stem Cells Using Mutantc-Myc Preparation of the Human Transcriptional Factor c-Myc with SilentMutations Introduced (Hereinafter Referred to as “c-rMyc”)

PCR was carried out (94° C. for three minutes, and 25 cycles of [98° C.for 30 seconds, 55° C. for 30 seconds, and 72° C. for six minutes],followed by 72° C. for seven minutes) using pBS-KS-c-Myc as a template,together with PrimeStar HS DNA polymerase (Takara Bio, catalog No.R010A) and the following six primers for mutagenesis: (c-rMyc1-F(5′-CGGACGACGAGACCTTCATCAAGAACATCATCATCCAGGACTG-3′ (SEQ ID NO: 39)),c-rMyc1-R (5′-CAGTCCTGGATGATGATGTTCTTGATGAAGGTCTCGTCGTCCG-3′ (SEQ ID NO:40)), c-rMyc2-F (5′-GAACGAGCTAAAACGGAGCTTCTTCGCCCTGCGTGACCAGATCC-3′ (SEQID NO: 41)), c-rMyc2-R(5T-GGATCTGGTCACGCAGGGCGAAGAAGCTCCGTTTTAGCTCGTTC-3′ (SEQ ID NO: 42)),c-rMyc3-F (5′-CCCAAGGTAGTTATCCTTAAGAAGGCCACAGCATACATCCTGTC-3′ (SEQ IDNO: 43)), and c-rMyc3-R(5′-GACAGGATGTATGCTGTGGCCTTCTTAAGGATAACTACCTTGGG-3′ (SEQ ID NO: 44))).The PCR product was treated with DpnI at 37° C. for two hours. E. coliDH5α (ToYoBo, Code No. DNA-903) was transformed with 5 μl of thereaction mixture. 16 E. coli colonies were isolated and mini-prep wasperformed. A clone that has the correct sequence was selected bysequencing, and thus pBS-KS-c-rMyc was obtained. pBS-KS-c-rMyc wasdigested with NotI at 37° C. for three hours, and separated byelectrophoresis using 1% agarose gel. A band of about 1.5 kbp wasexcised, and the DNA was purified using a Qiaquick Gel Extraction Kit(QIAGEN, catalog No. 28706). The NotI fragment containing the c-rMycgene was cloned into the NotI site of the pSeV (HNL)/TSΔF vector. Aclone that has the correct sequence was selected by sequencing, and thuspSeV(HNL)-c-rMyc/TSΔF was obtained. The nucleotide and amino acidsequences of c-rMyc are shown in SEQ ID NOs: 45 and 46, respectively.c-rMyc has the a378g, t1122c, t1125c, a1191g, and a1194g mutations.

pSeV(HNL)/TSΔF was constructed as follows. PCR was carried out (94° C.for one minute, and 30 cycles of [94° C. for 30 seconds, 55° C. for oneminute, and 68° C. for 22 minutes], followed by 68° C. for sevenminutes) using Litmus SalINheIfrg PmutMtsHNts ΔF-GFP (InternationalPublication No. WO2003/025570) as a template, together with thefollowing primers:

del GFP-Pac F (5′-GAGGTCGCGCGTTAATTAAGCTTTCACCTCAAACAAGCACAGATCATGG-3′(SEQ ID NO: 47)) and del GFP-Pac R(5′-GCATGTTTCCCAAGGGGAGAGTTAATTAACCAAGCACTCACAAGGGAC-3′ (SEQ ID NO:48)). The PCR product was treated in succession with Pad and DpnI. Theresulting product was self-ligated. A plasmid that has the correctsequence without the GFP gene was selected by sequencing, and thusLitmus SalINheIfrg PmutMtsHNts ΔF-GFP delGFP was obtained. PCR wascarried out (94° C. for three minutes, and 25 cycles of [98° C. for 10seconds, 55° C. for 15 seconds, and 72° C. for 12 minutes], followed by72° C. for seven minutes) using Litmus SalINheIfrg PmutMtsHNts ΔF-GFPdelGFP as a template, together with the following primers:HNLNOTI-F:5′-GGGTGAATGGGAAGCGGCCGCTAGGTCATGGATGG-3′ (SEQ ID NO: 49) andHNLNOTI-R:5′-CCATCCATGACCTAGCGGCCGCTTCCCATTCACCC-3′ (SEQ ID NO: 50). ThePCR product was digested with DpnI, and then E. coli DH5a (ToYoBo, CodeNo. DNA-903) was transformed with 20 μl of the reaction mixture. Six E.coli colonies were isolated and mini-prep was performed. A plasmid thathas the inserted NotI sequence was selected by NotI digestion. Then, aclone that has the correct sequence was selected by sequencing. Thus,Litmus SalINheIfrg PmutMtsHNts(HNL)-dF was obtained. Then, LitmusSalINheIfrg PmutMtsHNts(HNL)-dF was digested with SalI and NheI. Theresulting fragment was ligated to a fragment prepared by SalI/NheIdigestion of the pSeV/ΔSalINheIfrg Lmut plasmid (InternationalPublication No. WO2003/025570) whose L gene has two mutations. Thus,pSeV (HNL)/TSΔF was obtained. This vector encodes the same viralproteins as SeV18+/TSΔF, and has an insertion site (NotI site) for anintroduced gene between the HN and L genes.Preparation of a Sendai Virus Vector Carrying c-rMyc (theSeV(HNL)-c-rMyc/TSΔF Vector)

On the previous day of transfection, 10⁶ 293 T/17 cells were seeded intoeach well of a 6-well plate, and cultured in a CO₂ incubator (5% CO₂) at37° C. Using 15 μl of TransIT-LT1 (Mirus), the 293T/17 cells weretransfected with a mixture of: 0.5 μs of pCAGGS-NP, 0.5 μg of pCAGGS-P4C(−), 2 μg of pCAGGS-L (TDK), 0.5 μg of pCAGGS-T7, 0.5 μg of pCAGGS-F5R,and 0.5 μg of the Sendai virus vector plasmid pSeV(HNL)-c-rMyc/TSΔFdescribed above that carries the human transcriptional factor c-rMyc.The cells were cultured in a CO₂ incubator at 37° C. for two days. Then,10⁶ LLC-MK2/F/A cells which express the fusion protein (F protein) ofSendai virus were overlaid onto the transfected 293T/17 cells in eachwell, and the cells were cultured in a CO₂ incubator at 37° C. for oneday. On the following day, the cell culture medium was removed, and thecells were washed once with 1 ml of MEM supplemented withpenicillin-streptomycin (hereinafter abbreviated as PS/MEM). 1 nil ofPS/MEM containing 2.5 μg/ml trypsin (hereinafter abbreviated asTry/PS/MEM) was added to each well. The cells were cultured in a CO₂incubator at 32° C. for two days. The cells were continuously culturedwhile exchanging the medium every three to four days, and in some cases,passaging with LLC-MK2/F/A cells. An aliquot of the culture supernatantwas assessed for vector collection by hemagglutination assay. Theculture supernatant was harvested after sufficient hemagglutination wasobserved. RNA was extracted from the harvested culture supernatant usinga QIAamp Viral RNA Mini Kit (QIAGEN, catalog No. 52906), and subjectedto RT-PCR that targets a region of inserted c-rMyc. Whether the obtainedRT-PCR product has the correct nucleotide sequence was confirmed bysequencing. Thus, the SeV(HNL)-c-rMyc/TSΔF vector was obtained.

Example 5 iPS Induction Efficiency of Sendai Virus Vectors CarryingReprogramming Factors

The iPS induction efficiency of Sendai virus vectors carryingreprogramming factors is shown in the Table. The number of ES-likecolonies formed is shown along with the number of Sendai virus-infectedcells overlaid onto feeder cells. The experiment was carried out asdescribed in Example 1, except that the above c-rMyc-carrying vector wasadditionally used.

Of the reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc, modifiedc-Myc (c-rMyc) maximized the number of colonies formed, when it wasinserted into the HNL site of the vector. The induction efficiency wasabout ten times greater than that achieved by using retroviral vectors.Meanwhile, even when the three factors excluding Myc were used, iPSinduction was possible utilizing the Sendai virus vectors. Furthermore,using the Sendai virus vectors, iPS cells could be induced not only fromhuman newborn foreskin-derived cells (BJ) but also from human adultskin-derived cells (HDF) with an efficiency comparable to that of BJ.This result demonstrates that the methods of the present invention,which are simpler than conventional methods, allow high efficiency iPScell induction.

TABLE 1 Number of Parental Number of ES-like strain Origin cells seededcolonies c-Myc BJ Human newborn 5 × 10⁵ 14 Wild type foreskin HDF Humanadult 5 × 10⁵ 25 Wild type skin (face) BJ 5 × 10⁴ 28 Wild type BJ 3.5 ×10⁴   58 Wild type BJ 3.5 × 10⁴   67 HNL-rMyc BJ 5 × 10⁴ 118 HNL-rMyc BJ3.5 × 10⁵   6 Without Myc “Wild type” indicates the SeV18 + c-Myc/TSΔFvector carrying the wild-type c-Myc gene. “HNL-rMyc” indicates thepSeV(HNL)-c-rMyc/TSΔF vector carrying the silent mutant c-rMyc genebetween the HN and L genes, which is described in Example 4.

Example 6 ES Marker Expression in iPS Cells

iPS cells induced by the Sendai virus vectors carrying reprogrammingfactors were assessed for ES marker expression. iPS cells were inducedas described in Example 1, except that the above c-rMyc-carrying vectorwas additionally used. ES cell-like colonies were isolated using a stemcell knife (NIPPON MEDICAL & CHEMICAL INSTRUMENTS CO.) under amicroscope, and then passaged. RNA was extracted from each strain, andRT reaction and PCR were carried out in the same way as in FIG. 3.RT-PCR was performed to assess the expression of ES cell markers such asOct3/4, Nanog, Tert, and the following eight genes: GDF3, TDGF1, Zfp42,Sal4F, Dmmt3b, CABRB3, CYP26A1, and FoxD3 (Adewumi, O. et al.,Characterization of human embryonic stem cell lines by the InternationalStem Cell Initiative. Nat. Biotechnol. 25, 803-816, 2007), and theexpression of the reprogramming factors, Sox2, Klf4, and c-Myc. Themethod is as described in Example 3. It was demonstrated that all fiveclones expressed all of the markers (FIG. 4).

The primers used are listed below:

TERT F2847  (TGCCCGGACCTCCATCAGAGCCAG (SEQ ID NO: 37))  and  TERT R3399(TCAGTCCAGGATGGTCTTGAAGTCTG (SEQ ID NO: 38)) for  TERT; GDF3 F (GGCGTCCGCGGGAATGTACTTC (SEQ ID NO: 51))  and  GDF3 R(TGGCTTAGGGGTGGTCTGGCC (SEQ ID NO: 52) for GDF3; TDGF1-F1 (ATGGACTGCAGGAAGATGGCCCGC (SEQ ID NO: 53))  and  TDGF1-R567(TTAATAGTAGCTTTGTATAGAAAGGC (SEQ ID NO: 54)) for TDGF1; Zfp42-F1 (ATGAGCCAGCAACTGAAGAAACGGGCAAAG (SEQ ID NO: 55))  and Zfp42-R933 (CTACTTTCCCTCTTGTTCATTCTTGTTCG (SEQ ID NO: 56))  for Zfp42; SalI4 F (AAACCCCAGCACATCAACTC (SEQ ID NO: 57))  and  SalI4 R(GTCATTCCCTGGGTGGTTC (SEQ ID NO: 58)) for SalI4; Dnmt3b F (GCAGCGACCAGTCCTCCGACT (SEQ ID NO: 59))  and  Dnmt3b R(AACGTGGGGAAGGCCTGTGC (SEQ ID NO: 60)) for Dmmt3b; GABRB3 F (CTTGACAATCGAGTGGCTGA (SEQ ID NO: 61))  and  GABRB3 R(TCATCCGTGGTGTAGCCATA (SEQ ID NO: 62)) for GABRB3; CYP26A1 F (AACCTGCACGACTCCTCGCACA (SEQ ID NO: 63))  and  CYP26A1 R(AGGATGCGCATGGCGATTCG (SEQ ID NO: 64)) for  CYP26A1; FoxD3-F418 (GTGAAGCCGCCTTACTCGTAC (SEQ ID NO: 65))  and  FoxD3-R770(CCGAAGCTCTGCATCATGAG (SEQ ID NO: 66)) for FOXD3; F6 (ACAAGAGAAAAAACATGTATGG (SEQ ID NO: 67))  and  OCT3/4 R259(GAGAGGTCTCCAAGCCGCCTTGG (SEQ ID NO: 68)) for  SeV-Oct3/4; Sox2-F294 (AGCGCTGCACATGAAGGAGCACC (SEQ ID NO: 27))  and  R150(AATGTATCGAAGGTGCTCAA (SEQ ID NO: 69)) for  SeV-Sox2; F6 (ACAAGAGAAAAAACATGTATGG (SEQ ID NO: 67)) and  KIF4-R405(CGCGCTGGCAGGGCCGCTGCTCGAC (SEQ ID NO: 70))  for SeV-Klf4; F6 (ACAAGAGAAAAAACATGTATGG (SEQ ID NO: 67))  and  c-rMyc406(TCCACATACAGTCCTGGATGATGATG (SEQ ID NO: 71)) for  SeV-c-Myc; and F8424 (TAACTGACIAGCAGGCTTGTCG (SEQ ID NO: 72))  and  c-rMyc406(TCCACATACAGTCCTGGATGATGATG (SEQ ID NO: 71))  for c-Myc/HNL.

Example 7 Telomerase Activity of iPS Cells

Telomerase activity was assayed to assess the ability of infiniteproliferation of the iPS cells induced with the Sendai virus vectorscarrying reprogramming factors. iPS cells were induced as described inExample 1, except that the above c-rMyc-carrying vector (referred to asHNL) was additionally used. A TRAPEZE™ Telomerase Detection Kit(CHEMICON, Cat. No. S7700) was used to detect the telomerase activity.The cells were harvested, and 200 μl of 1×CAPS lysis buffer attached tothe kit was added thereto. The cells were suspended by pipetting. Thiswas incubated on ice for 30 minutes, and then centrifuged in arefrigerated microfuge at 12,000 rpm and 4° C. for 20 minutes. 160 μl ofthe supernatant was transferred to another Eppendorf tube, and this celllysate was assessed for its protein concentration. Before the assay, analiquot of the cell lysate including 1 μg of protein was placed into anEppendorf tube and heated at 85° C. for ten minutes. 1 μg each of theheat-treated and non-treated samples was used for TRAP assay. For eachassay, a reaction mixture was prepared by combining the following: 5.0μl of 10×TRAP reaction buffer, 1.0 μl of 50×dNTP mix, 1.0 μl of TSprimer, 1.0 μl of TRAP primer mix, 40.6 μl of [cell lysate (1 μg ofprotein) and water], and 0.4 μl of Taq polymerase. PCR was carried outas follows: 30° C. for 30 minutes, followed by 30 cycles of [94° C. for30 seconds, 59° C. for 30 seconds, and 72° C. for 60 seconds]. 6×loading dye was added to the PCR reaction mixture. 20 μl of this wasloaded onto 10% or 12.5% polyacrylamide gel. The gel afterelectrophoresis was stained with ethidium bromide.

All iPS clones exhibited telomerase activity. The activity was notdetected in the parental BJ and HDF lines which are controls, andheat-treated iPS cells (FIG. 5).

Example 8 Pluripotency of iPS Cells

An in vitro embryoid body formation experiment was conducted to assessthe pluripotency of iPS cells induced with the Sendai virus vectorscarrying reprogramming factors. iPS cells were induced as described inExample 1. Colonies of three iPS clones, 4BJ1, B1 (derived from BJ), and7H5 (derived from HDF), were detached from dishes using collagenase IV(Invitrogen, 17104-019). The cell masses were transferred intoMPC-coated wells (Nunc, 145383) and incubated for several days insuspension culture in RPMI 1640 supplemented with 10% FBS. Embryoid bodyformation was observed under a microscope. iPS cells induced with theSendai virus vectors had differentiation ability, and all the iPS cellsformed embryoid bodies. Many cystic embryoid bodies at a moredifferentiated stage were observed on day seven (FIG. 6).

Furthermore, to show the pluripotency for triploblastic differentiationof iPS cells induced with the Sendai virus vectors (SeV-iPS), the cellswere induced in vitro for differentiation into cardiac muscle cells(mesoderm), dopamine-producing neurons (ectoderm), and pancreatic cells(endoderm). SeV-iPS clones from which SeV vectors were removed weredetached from feeder cells using 1 mg/ml collagenase IV. For cardiacmuscle cell induction, the cells were incubated for six days insuspension culture in NPC-coated plates containing DMEM supplementedwith 20% FBS and 0.1 mM vitamin C. After formation of embryoid bodies,they were transferred into plates coated with 0.1% gelatin, and culturedfor one week. Thus, pulsing cardiac muscle was obtained (Takahashi, T.et. al., Circulation 107, 1912-1916, 2003). For dopamine-producingneuron induction, iPS cells were isolated in the same manner, and seededonto confluent PA6 feeder cells (RIKEN BRC) in 0.1% gelatin-coatedplates. The cells were cultured for 16 days in GMEM (Invitrogen)supplemented with 10% KSR, 2 mM L-glutamine and nonessential aminoacids, and 2-mercaptoethanol at a final concentration of 1×10⁻⁴M. Afterfixation with 10% formalin solution, the cells were stained with ananti-βIII tubulin antibody (SantaCruz; 2G10) and an anti-tyrosinehydroxilase antibody (Chemicon; P07101) to assess whether they aredopamine-producing neurons (Kawasaki, H. et al., Neuron 28, 31-40(2000)). For pancreatic cell induction, SeV-iPS cells were cultured forfour days on MMC-treated MEF feeder cells in RPMI 1640 supplemented with2% FBS and 100 ng/ml activin A (R&D Systems). Then, the cells furthercultured for eight days in DMEM/F, 12 medium supplemented with N2 andB27 supplements, 2 mM L-glutamine and nonessential amino acids, 1×10⁻⁴M2-mercaptoethanol, and 0.5 mg/ml BSA (Invitrogen). The cells were fixedwith 10% formalin solution, and stained with an anti-PDX antibody (R&DSystems; AF2419) and an anti-SOX17 antibody (R&D Systems; 245013) todetect pancreatic β cells and endodermal cells, respectively (D'Amour,K. A. et al., Nat. Biotechnol. 23, 1534-1541 (2005)) (FIG. 7).

Furthermore, the in vitro pluripotency was confirmed by teratomaformation in immunodeficient mice. SeV-iPS cells were subcutaneouslyinoculated into SCID mice. Tumor formation was observed after about onemonth. Then, after about two months, samples were collected and fixedwith 10% formalin, and embedded in paraffin. Tissue sections werestained with hematoxylin/eosin to assess the triploblasticdifferentiation (FIG. 8).

Example 9 Promoter Analysis of iPS Cells

To assess whether the Oct3/4 and Nanog gene promoters, which areexpressed in ES cells, are also activated in iPS cells, methylationanalysis was performed by the bisulfite sequencing method describedbelow. The result showed that the Oct3/4 promoter (region from −2330 to−2110) and Nanog promoter (region from −685 to −120) were highlydemethylated in each SeV-iPS cell clone, while the promoters were highlymethylated in the parental BJ and HDF lines as the controls. Thus, theOct3/4 and Nanog promoters were demonstrated to be activated in SeV-iPScells as in ES cells (FIG. 9).

(Bisulfite Sequencing Method)

Genomic DNA was extracted from iPS cells using a QIAamp DNA Mini Kit(50) (QIAGEN, catalog No. 51304) according to the protocol appended tothe kit. Then, 1 μg of the extracted genomic DNA was modified withbisulfite using a BisulFast DNA Modification Kit for Methylated DNADetection (Toyobo, catalog No. MDD-101) according to the attachedprotocol. PCR was carried out using the bisulfite-modified genomic DNAas a template, together with specific primers that target the promoterregions of the Oct3/4 and Nanog genes. The PCR product was separated byagarose gel electrophoresis. The bands of interest were purified using aQIAquick Gel Extraction Kit (QIAGEN, catalog No. 28704). The purifiedPCR product was TA-cloned using pGEM-T Easy Vector System I (Promega,catalog No. A1360) according to the attached protocol. Then, colony PCRwas carried out using specific primers that target the promoter regionsof the Oct3/4 and Nanog genes. About ten clones that gave a band of thecorrect size were selected by agarose gel electrophoresis. Plasmid DNAswere extracted from the clones by mini-prep, and sequenced using the T7and SP6 primers. Methylation of the promoter regions was assessed bycomparing the sequences with the target sequences after bisulfitemodification.

PCR primers for amplification of the Oct3/4 gene promoter region andcolony PCR (J. Biol. Chem., 2005, Vol. 280, 6257-6260):

mOct4-5F: 5′-AATAGATTTTGAAGGGGAGTTTAGG-3′ (SEQ ID NO: 73); and

mOct4-5R: 5′-TTCCTCCTTCCTCTAAAAAACTCA-3′ (SEQ ID NO: 74)

PCR primers for amplification of the Nanog gene promoter region andcolony PCR (Stem cell Research, Vol. 1, 105-115; Cell, 2007, Vol. 131,861-72):

Nanog-z1-L: 5′-GGAATTTAAGGTGTATGTATTTTTTATTTT-3′ (SEQ ID NO: 75); and

mehNANOG-F1-AS: 5′-AACCCACCCTTATAAATTCTCAATTA-3′ (SEQ ID NO: 76)

Sequencing Primers:

T7: 5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 77); and

SP6: 5′-CATACGATTTAGGTGACACTATAG-3′ (SEQ ID NO: 78)

Kit Used:

BisulFast DNA Modification Kit for Methylated DNA Detection (Toyobo,catalog No. MDD-101)

Example 10 Gene Expression Analysis of iPS Cells

The gene expression profile of iPS cells induced with the Sendai virusvectors (SeV-iPS cells) was compared to those of the parental BJ cellline, human ES cells, and previously established human iPS cells(GSM241846; Takahashi, K. et al., Cell, 131, 1-12, 2007). Total RNAswere extracted from SeV-iPS and BJ cells using an RNeasy Mini Kit(Qiagen). Cyanine dye-labeled cRNAs were synthesized from cDNAs using aQuick Amp Labeling Kit (Agilent). The cRNAs were hybridized with theWhole Human Genome Oligo Microarray (4×44K) for 17 hours using a GeneExpression Hybridization Kit (Agilent). After washing, the images of theDNA microarray were scanned using an Agilent Microarray Scanner.Fluorescent signal at each spot was digitized and analyzed by FeatureExtraction Software (v.9.5.3.1). A total of 41078 probes on the chip,excluding those overlapped, were analyzed (BIO MATRIX RESEARCH). Thegene expression in SeV-iPS cells was compared to the gene expression inthe following controls, whose information was obtained from GEODetaSets: human ES cells, hES-H9 (GSM194390; Teser P. J., et al., Nature448, 196-199, 2007), and human iPS cells, hiPS induced from HDF(GSM241846; Takahashi, K. et al., Cell, 131, 1-12, 2007). The resultshowed that the correlation of SeV-iPS with BJ was r=8732, with human EScells was r=0.9658, and with human iPS cells was r=0.9580. The Nanog,Sox2, and Oct3/4 genes, which are expressed in ES cells, were alsoexpressed in SeV-iPS. While there was no correlation with BJ, theprofiles of SeV-iPS and human ES cells or human iPS cells were locatedon the correlation line, and they completely matched (FIG. 9).

Example 11 Preparation of Vectors into which Temperature-DependentInactivation Mutations are Introduced

(Method for Preparing Vectors)

Construction of Plasmids for Preparing Sendai Virus Vectors into whichTemperature-Dependent Inactivation Mutations are Introduced

PCR was carried out (94° C. for three minutes, and 25 cycles of [98° C.for 10 seconds, 55° C. for 15 seconds, and 72° C. for 11 minutes],followed by 72° C. for seven minutes) using Litmus SalINheIfrgPmutMtsHNts ΔF-GFP (WO2003/025570) as a template, together with thefollowing:

the combination of: L Y942H-F(5′-CAAATGTTGGAGGATTCAACCACATGTCTACATCTAGATG-3′ (SEQ ID NO: 79)) and LY942H-R (5′-CATCTAGATGTAGACATGTGGTTGAATCCTCCAACATTTG-3′ (SEQ ID NO:80)); and

the combination of: L Y942H-F, L Y942H-R, P2-F(5′-CATCACAGCTGCAGGTGGCGCGACTGACAAC-3′ (SEQ ID NO: 81)), and P2-R(5′-GTTGTCAGTCGCGCCACCTGCAGCTGTGATG-3′ (SEQ ID NO: 82)). The PCRproducts were digested with DpnI at 37° C. for one hour. E. coli DH5α(ToYoBo, Code No. DNA-903) was transformed with 20 μl of the reactionmixture. Colonies formed were isolated and mini-prep was performed.Then, clones that have the correct sequences were selected bysequencing, and thus Litmus38TSΔF-GFP-LY942H andLitmus38TSΔF-GFP-P2LY942H were obtained.

Litmus38TSΔF-GFP-P2LY942H was digested with StuI, and then this wasseparated by agarose gel electrophoresis. A band of 1.9 kbp was excised,and the DNA was purified. Litmus SalINheIfrg PmutMtsHNts ΔF-GFP wasdigested with StuI, and then this was separated by agarose gelelectrophoresis. A band of 9.8 kbp was excised, and the DNA waspurified. The two purified fragments were ligated together to constructLitmus38TSΔF-GFP-P2.

Litmus38TSΔF-GFP-P2LY942H was digested with NcoI, and then this wasseparated by agarose gel electrophoresis. A band of 7.1 kbp was excised,and the DNA was purified. Litmus SalINheIfrg PmutMtsHNts ΔF-GFP delGFPwas digested with NcoI, and then this was separated by agarose gelelectrophoresis. A band of 3.7 kbp was excised, and the DNA waspurified. The purified DNAs were ligated together. The structure of theproduct was confirmed by colony PCR and double digestion with NcoI andPacI. Thus, Litmus38TSΔF-P2LY942IIΔGFP was obtained.

pSeV(HNL)/TSΔF was digested with NcoI, and then this was separated byagarose gel electrophoresis. A band of 3.7 kbp was excised, and the DNAwas purified. The resulting fragment was ligated to the above 7.1-kbpNcoI fragment from Litmus38TSΔF-GFP-P2LY942H to prepareLitmus38TSΔF-P2LY942H(HNL)ΔGFP.

Litmus38TSΔF-GFP-P2 was digested with NcoI, and then this was separatedby agarose gel electrophoresis. A band of 7.1 kbp was excised, and theDNA was purified. The resulting fragment was ligated to the aboveNcoI-digested and purified fragment (3.7 kbp) from Litmus SalINheIfrgPmutMtsHNts ΔF-GFP delGFP and NcoI-digested and purified fragment (3.7kbp) from pSeV(HNL)/TSΔF to construct Litmus38TSΔF-P2ΔGFP andLitmus38TSΔF-P2(HNL)ΔGFP, respectively.

PCR was carried out (94° C. for three minutes, and 25 cycles of [98° C.for 10 seconds, 55° C. for 15 seconds, and 72° C. for nine minutes],followed by 72° C. for 7 minutes) using pSeV/ΔSalINheIfrg Lmut(WO2003/025570) as a template, together with the following: thecombination of: L L1361C-F(5′-GGTTCCTTAGGGAAGCCATGTATATTGCACTTACATCTTA-3′ (SEQ ID NO: 83)) and LL1361C-R (5′-TAAGATGTAAGTGCAATATACATGGCTTCCCTAAGGAACC-3′ (SEQ ID NO:84));

the combination of: L L1558I-F(5′-CCTGTGTATGGGCCTAACATCTCAAATCAGGATAAGATAC-3′ (SEQ ID NO: 85)) and LL1558I-R (5′-GTATCTTATCCTGATTTGAGATGTTAGGCCCATACACAGG-3′ (SEQ ID NO:86)); and

the combination of L L1361C-F, L L1361C-R, L L1558I-F, and L L1558I-R.The PCR products were digested with DpnI at 37° C. for one hour. E. coliDH5α (ToYoBo, Code No. DNA-903) was transformed with 20 μl of thereaction mixture. Colonies formed were isolated and mini-prep wasperformed. Clones that have the correct sequences were selected bysequencing, and thus pSeV/TSΔF-Linker L1361C, pSeV/TSΔF-Linker L1558I,and pSeV/TSΔF-Linker L1361CL1558I were obtained.

Litmus38TSΔF-P2LY942H(HNL)ΔGFP and pSeV/TSΔF-Linker L1361CL1558I wereeach digested with SalI and NheI, and then the digests were separated byagarose gel electrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively,were excised, and the DNAs were purified. The purified fragments wereligated together to construct pSeV(HNL)/TS8ΔF.

pSeV(HNL)/TS8ΔF and pSeV(HNL)/TSΔF were digested with NotI and XhoI, andthe digests were separated by agarose gel electrophoresis. Bands of 4.9kbp and 11.4 kbp, respectively, were excised, and the DNAs werepurified. The purified fragments were ligated together to constructpSeV(HNL)/TS7ΔF. pBS-KS-c-rMyc was digested with NotI. The resultingNotI fragment containing the c-rMyc gene was excised and purified, andthen this was inserted into the NotI site of the pSeV(HNL)/TS7ΔF vectorto construct pSeV(HNL)-c-rMyc/TS7ΔF.

Litmus38TSΔF-P2LY942HΔGFP and pSeV/TSΔF-Linker L1361CL1558I were eachdigested with SalI and NheI, and then the digests were separated byagarose gel electrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively,were excised, and the DNAs were purified. The purified fragments wereligated together to construct pSeV18+BSSHII/TS8ΔF. pSeV18+BSSHII/TS8ΔFand pSeV18+Oct3/4/TSΔF were each digested with AatII and SphI, and bandsof 15.2 kbp and 2.3 kbp, respectively, were excised, and the DNAs werepurified. The purified fragments were ligated together to constructpSeV18+Oct3/4/TS8ΔF. pSeV18+Oct3/4/TS8ΔF and pSeV18+/TSΔF were eachdigested with PacI and SphI, and then the digests were separated byagarose gel electrophoresis. Bands of 13.3 kbp and 4.2 kbp,respectively, were excised, and the DNAs were purified. The purifiedfragments were ligated together to construct pSeV18+Oct3/4/TS7ΔF. Then,pSeV18+Oct3/4/TS7ΔF was digested with NotI, and then this was separatedby agarose gel electrophoresis. A band of 16.4 kbp was excised, and theDNA was purified. The purified fragment was ligated to the NotIfragments each containing the Sox2, KLF4, or c-rMyc gene, which wereexcised by NotI digestion from pBS-KS-Sox2, pBS-KS-KLF4, andpBS-KS-c-rMyc described above, respectively, and then purified. Thus,pSeV18+Sox2/TS7ΔF, pSeV18+KLF4/TS7ΔF, and pSeV18+c-rMyc/TS7ΔF wereobtained.

Litmus38TSΔF-P2(HNL)ΔGFP and pSeV/TSΔF-Linker L1361C were each digestedwith SalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV (HNL)/TS14ΔF. pBS-KS-c-rMyc was digested withNotI. The resulting NotI fragment containing the c-rMyc gene was excisedand purified, and then this was inserted into the NotI site ofpSeV(HNL)/TS14ΔF to construct pSeV(HNL)-c-rMyc/TS14ΔF.

Litmus38TSΔF-P2(HNL)ΔGFP and pSeV/TSΔF-Linker L1558I were each digestedwith SalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV(HNL)/TS13ΔF. pBS-KS-c-rMyc was digested withNotI. The resulting NotI fragment containing the c-rMyc gene was excisedand purified, and then this was inserted into the NotI site ofpSeV(HNL)/TS13ΔF to construct pSeV(HNL)-c-rMyc/TS13ΔF.

Litmus38TSΔF-P2(HNL)ΔGFP and pSeV/TSΔF-Linker L1361CL1558I were eachdigested with SalI and NheI, and then the digests were separated byagarose gel electrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively,were excised, and the DNAs were purified. The purified fragments wereligated together to construct pSeV(HNL)/TS15ΔF. pBS-KS-c-rMyc wasdigested with NotI. The resulting NotI fragment containing the c-rMycgene was excised and purified, and then this was inserted into the NotIsite of pSeV(HNL)/TS15ΔF to construct pSeV(HNL)-c-rMyc/TS15ΔF.

Litmus38TSΔF-P2ΔGFP and pSeV/TSΔF-Linker L1361C were each digested withSalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV18+BSSHII/TS14ΔF. pSeV18+BSSHII/TS14ΔF wasdigested with AatII and SphI, and a band of 15.2 kbp was excised, andthe DNA was purified. The purified fragment was ligated to the aboveAatII-SphI fragment (2.3 kbp) from pSeV18+Oct3/4/TSΔF to constructpSeV18+Oct3/4/TS14ΔF. Then, pSeV18+Oct3/4/TS14ΔF was digested with NotI,and then this was separated by agarose gel electrophoresis. A band of16.4 kbp was excised, and the DNA was purified. The purified fragmentwas ligated to the NotI fragments each containing the Sox2, KLF4, orc-rMyc gene, which were excised by NotI digestion from pBS-KS-Sox2,pBS-KS-KLF4, and pBS-KS-c-rMyc described above, respectively, and thenpurified. Thus, pSeV18+Sox2/TS14ΔF, pSeV18+KLF4/TS14ΔF, andpSeV18+c-rMyc/TS14ΔF were obtained.

Litmus38TSΔF-P2ΔGFP and pSeV/TSΔF-Linker L1558I were each digested withSalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV18+BSSHII/TS13ΔF. pSeV18+BSSHII/TS13ΔF wasdigested with AatII and SphI, and a band of 15.2 kbp was excised, andthe DNA was purified. The purified fragment was ligated to the aboveAatII-SphI fragment (2.3 kbp) from pSeV18+Oct3/4/TSΔF to constructpSeV18+Oct3/4/TS13ΔF. Then, pSeV18+Oct3/4/TS13ΔF was digested with NotI,and then this was separated by agarose gel electrophoresis. A band of16.4 kbp was excised, and the DNA was purified. The purified fragmentwas ligated to the above NotI fragments each containing the Sox2, KLF4,or c-rMyc gene. Thus, pSeV18+Sox2/TS13ΔF, pSeV18+KLF4/TS13ΔF, andpSeV18+c-rMyc/TS13ΔF were obtained.

Litmus38TSΔF-P2ΔGFP and pSeV/TS ΔF-Linker L1361CL1558I were eachdigested with SalI and NheI, and then the digests were separated byagarose gel electrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively,were excised, and the DNAs were purified. The purified fragments wereligated together to construct pSeV18+BSSHII/TS15ΔF. pSeV18+BSSHII/TS15ΔFwas digested with AatII and SphI, and a band of 15.2 kbp was excised,and the DNA was purified. The purified fragment was ligated to the aboveAatII-SphI fragment (2.3 kbp) from pSeV18+Oct3/4/TSΔF to constructpSeV18+Oct3/4/TS15ΔF. Then, pSeV18+Oct3/4/TS15ΔF was digested with NotI,and then this was separated by agarose gel electrophoresis. A band of16.4 kbp was excised, and the DNA was purified. The purified fragmentwas ligated to the above NotI fragments each containing the Sox2, KLF4,or c-rMyc gene. Thus, pSeV18+Sox2/TS15ΔF, pSeV18+KLF4/TS15ΔF, andpSeV18+c-rMyc/TS15ΔF were obtained.

Litmus38TSΔF-P2(HNL)ΔGFP and pSeV/ΔSalINheIfrg Lmut were each digestedwith SalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV (HNL)/TS12ΔF. pBS-KS-c-rMyc was digested withNotI. The resulting NotI fragment containing the c-rMyc gene was excisedand purified, and then this was inserted into the NotI site ofpSeV(HNL)/TS12ΔF to construct pSeV(IINL)-c-rMyc/TS12ΔF.

Litmus38TSΔF-P2ΔGFP and pSeV/ΔSalINheIfrg Lmut were each digested withSalI and NheI, and then the digests were separated by agarose gelelectrophoresis. Bands of 8.0 kbp and 8.3 kbp, respectively, wereexcised, and the DNAs were purified. The purified fragments were ligatedtogether to construct pSeV18+BSSHII/TS12ΔF. pSeV18+BSSHII/TS12ΔF wasdigested with AatII and SphI, and a band of 15.2 kbp was excised, andthe DNA was purified. The purified fragment was ligated to the aboveAatII-SphI fragment (2.3 kbp) from pSeV18+Oct3/4/TSΔF to constructpSeV18+Oct3/4/TS12ΔF. Then, pSeV18+Oct3/4/TS12ΔF was digested with NotI,and then this was separated by agarose gel electrophoresis. A band of16.4 kbp was excised, and the DNA was purified. The purified fragmentwas ligated to the above NotI fragments each containing the Sox2, KLF4,or c-rMyc gene. Thus, pSeV18+Sox2/TS12ΔF, pSeV18+KLF4/TS12ΔF, andpSeV18+c-rMyc/TS12ΔF were obtained.

(Collection of F Gene-Deficient Sendai Virus Vectors into whichTemperature-Dependent Inactivation Mutations are Introduced)

On the previous day of transfection, 10⁶ 293 T/17 cells were seeded intoeach well of a 6-well plate, and cultured in a CO₂ incubator (5% CO₂) at37° C. Using 15 μl of TransIT-LT1 (Mirus), the 293T/17 cells weretransfected with a mixture of: 0.5 μg of pCAGGS-NP, 0.5 μg of pCAGGS-P4C(−), 2 μg of pCAGGS-L (TDK), 0.5 μg of pCAGGS-T7, 0.5 μg of pCAGGS-F5R(WO2005/071085), and 0.5 μg of the above F gene-deficient Sendai virusvector plasmid into which temperature-dependent inactivation mutants areintroduced, and that carries a human transcriptional factor. The cellswere cultured in a CO₂ incubator at 37° C. for two to three days. Then,10⁶ LLC-MK2/F/A cells which express the fusion protein (F protein) ofSendai virus were overlaid onto the transfected 293T/17 cells in eachwell, and the cells were cultured in a CO₂ incubator at 37° C. for oneday. On the following day, the cell culture medium was removed, and thecells were washed once with 1 ml of MEM supplemented withpenicillin-streptomycin (hereinafter abbreviated as PS/MEM). 1 ml ofPS/MEM containing 2.5 μg/ml trypsin (hereinafter abbreviated asTry/PS/MEM) was added to each well. The cells were cultured in a CO₂incubator at 32° C. The cells were continuously cultured whileexchanging the medium every three to four days, and in some cases,passaging with LLC-MK2/F/A cells. An aliquot of the culture supernatantwas assessed for vector collection by hemagglutination assay. Theculture supernatant was harvested after sufficient hemagglutination wasobserved. RNA was extracted from the harvested culture supernatant usinga QIAamp Viral RNA Mini Kit (QIAGEN, catalog No. 52906), and subjectedto RT-PCR that targets a region of the inserted gene. Whether theobtained RT-PCR product has the correct nucleotide sequence wasconfirmed by sequencing. Thus, F gene-deficient Sendai virus vectorsinto which temperature-dependent inactivation mutations are introduced,and which carry various human transcriptional factors, were obtained.

Example 12 Vector Removal

Colonies in which a SeV vector was naturally removed from SeV-iPS cells,were obtained. Furthermore, Sendai virus-free clones were obtained bytemperature shift to 39° C. after induction of iPS at 37° C. using thetemperature-sensitive vectors. SeV-free clones were also obtained bynegative selection with an anti-HN antibody using as an indicator the HNantigen, which is expressed on the cell surface upon SeV infection.

1. Natural Removal

Passage culture of SeV-iPS cells led to an increase in the number ofcells from which the vectors were naturally removed. RNA was extractedfrom cells of the SeV-iPS colonies. RT-PCR was carried out to assess theexpression of foreign genes derived from SeV. When SeV-Oct3/4, Sox2,Klf4, and c-Myc (c-rMyc or c-Myc) were inserted at position 18+(the 3′end of the NP gene that is located at the most 3′ end of the genome)(SeV18+Oct3/4/TSΔF, SeV18+Sox2/TSΔF, SeV18+Klf4/TSΔF, andSeV18+c-Myc/TSΔF (or SeV18+c-rMyc/TSΔF), respectively), the foreigngenes were diluted via cell division, and often, only one or two of thegenes remained to be expressed. Wild-type c-Myc was eliminated first dueto its lower replicability. On the other hand, when c-rMyc was insertedat the position of HNL (HNL-c-rMyc/TSΔF), the cells often retained theMyc gene-carrying vector alone due to its higher replicability than thevectors into which a factor of interest was inserted at position 18+.Even clones from which all of the introduced foreign reprogrammingfactors were completely removed were obtained. The complete removal wasdemonstrated not only by RT-PCR but also at the protein level by Westernblotting using an anti-SeV-NP antibody (FIG. 10). The RT-PCR primersused are as described in Example 6.

2. Removal with an Anti-HN Antibody

SeV vectors are naturally removed by dilution via cell division andpassaging. Alternatively, SeV vector-free cells can be activelycollected. Utilizing an anti-HN antibody, SeV vector-removed cells canbe obtained using as an indicator the HN antigen, which is expressed onthe cell surface upon SeV infection. Cells were disaggregated into smallpopulations by collagenase IV and trypsin treatment and suspensionprocedure. The cells were reacted with the anti-HN monoclonal antibodyIL4.1 on ice for 30 minutes. After washing with medium, the cells werereacted with a secondary antibody, for example, an anti-mouse IgG1antibody bound to magnetic beads (Anti-Mouse IgG1 Particles; BD) on icefor 30 minutes. The unbound fraction was collected using a magnet(IMagnet Cell Separation Magnet; BD) (negative selection). Thus, a cellpopulation with impaired SeV vector expression was obtained. Vector-freeiPS cells were isolated by repeating the above treatments (FIG. 11).Alternatively, an anti-IIN antibody-negative cell population can beisolated by FACS.

3. Temperature-Sensitive Vector-Based Technique for SeV Removal

TS 7: L (Y942H/L1361C/L1558I)

TS 13: P (D433A/R434A/K437A), L (L1558I)

TS 14: P (D433A/R434A/K437A), L (L1361C)

TS 15: P (D433A/R434A/K437A), L (L1361C/L1558I)

The above mutations were introduced into the SeV18+/TSΔF vector. Theresulting vectors are temperature-sensitive, and their replication isinhibited by temperature shift. Specifically, an inserted gene isexpressed at the highest level at 32° C., and also expressed at 35 to36° C., and expressed at a slightly lower level at 37° C., but notexpressed at 38.5 or 39° C.

The reprogramming factors were inserted into these vectors in the samemanner as described above. iPS cells were induced at 37° C., and thetemperature was shifted after production of the iPS cells, and SeV couldbe readily removed from the cells.

4. Higher Replicability of HNL-Myc

As described in section 1, when SeV-18+Oct3/4, Klf4, Sox2, andSeV-HNL-c-rMyc were used in combination to induce iPS, SeV-HNL-c-rMyc inwhich the c-rMyc gene is inserted between HN and L was more advantageousin replication than the SeV vectors carrying the other factors insertedat position 18+(upstream of the NP gene). Furthermore, since c-Myc isbeneficial for cell growth, of the four factors inserted into SeV, onlySeV-HNL-c-rMyc was finally retained. SeV-iPS cells induced withSeV-HNL-c-rMyc were easily established as clones because they havesuperior proliferation ability. In addition, only one vector wasretained, and it tended to be naturally removed. Thus, only the vectorfor HNL-c-rMyc needs to be temperature-sensitive to achieve the removalby temperature shift using temperature-sensitive strains. In fact, theHNL-c-rMyc vector finally remained could be removed by temperature shift(FIGS. 12 and 13).

5. Preparation of iPS Cells from which Temperature-Sensitive SeV Vectorsare Readily Removable

When iPS cells are induced from fibroblasts (BJ cells) using the TSvectors each having Oct3/4, Klf4, or Sox2 as an insert at position 18+and the above-described vector (TS7ΔF, TS13ΔF, or TS15ΔF) having thec-rMyc gene as an insert between HN and L of TS7, TS13, or TS15, onlythe temperature-sensitive SeV-HNL-Myc is retained because of itssuperior replicability as described above. Furthermore, when the cellshave SeV-IINL-Myc alone, the last vector retained is rapidly eliminated,because the expression level of the temperature-sensitive strain islower at 37° C.

iPS was thus induced. The number of clones that became SeV vector-freewithin one month after induction was four out of six when usingTS/18+Oct3/4, Sox2, Klf4/TSΔF, and TS13ΔF/HNL-c-rMyc in combination;three out of six when using TS/18+Oct3/4, Sox2, Klf4/TSΔF, andTS15ΔF/HNL-c-rMyc in combination; and two out of twelve when using

TS/18+Oct3/4, Sox2, Klf4/TSΔF, and TS7ΔF/HNL-c-rMyc in combination (FIG.13). All of the obtained SeV-free clones expressed the human EScell-specific markers (FIG. 14).

This method allows simple preparation of SeV-free and intact iPS cellswithout damaging the chromosome.

Example 13

As described in section 5 of Example 12, iPS cells can be induced usingnot only the above-described TSΔF vectors but also TS7ΔF, TS13ΔF, andTS15ΔF into which the reprogramming factors (Oct3/4, Sox2, Klf4, andc-Myc) are inserted. Using an L mutant (Y1214F) with another ΔF vectorbackbone (WO2008/096811), whether iPS cells can also be induced in thesame manner was tested as follows.

(Construction of LmΔF/SeV)

Plasmid Construction

pSeV18+LacZ/ΔF-1214 (WO2008/096811) was digested with NotI, and this waspurified. Then, the resulting fragment was ligated, and a plasmidwithout the lacZ gene was selected. Thus, pSeV18+/ΔF-1214 (also referredto as “Lm (Y1214F) ΔF/SeV”, or simply, “LmΔF/SeV”) was obtained.

Next, pSeV18+/ΔF-1214 was digested with NotI, and this was purified.

The above-mentioned NotI fragments of the four reprogramming factorsOct3/4, Klf4, Sox2, and c-rMyc were each inserted into the above vectorto construct the plasmids pSeV18+Oct3/4/ΔF-1214, pSeV18+Sox2/ΔF-1214,pSeV18+KLF4/ΔF-1214, and pSeV18+c-rMyc/ΔF-1214 for preparation of viralvectors.

Collection of LmΔF/SeV Sendai Virus Vectors

On the previous day of transfection, 10⁶ 293 T/17 cells were seeded intoeach well of a 6-well plate, and cultured in a CO₂ incubator (5% CO₂) at37° C. Using 15 μl of TransIT-LT1 (Mirus), the 293T/17 cells weretransfected with a mixture of: 0.5 μg of pCAGGS-NP, 0.5 μg ofpCAGGS-P4C(−), 2 μg of pCAGGS-L (TDK), 0.5 μg of pCAGGS-T7, 0.5 μg ofpCAGGS-F5R (WO2005/071085), and 0.5 μg of an LmΔF/SeV Sendai virusvector plasmid carrying an above-described human transcriptional factor.The cells were cultured in a CO₂ incubator at 37° C. for two to threedays. Then, 10⁶ LLC-MK2/F/A cells which express the fusion protein (Fprotein) of Sendai virus were overlaid onto the transfected 293T/17cells in each well, and the cells were cultured in a CO₂ incubator at37° C. for one day. On the following day, the cell culture medium wasremoved, and the cells were washed once with 1 ml of MEM supplementedwith penicillin-streptomycin (hereinafter abbreviated as PS/MEM). 1 mlof PS/MEM containing 2.5 μg/ml trypsin (hereinafter abbreviated asTry/PS/MEM) was added to each well. The cells were cultured in a CO₂incubator at 32° C. The cells were continuously cultured whileexchanging the medium every three to four days, and in some cases,passaging with LLC-MK2/F/A cells. An aliquot of the culture supernatantwas assessed for vector collection by hemagglutination assay. Theculture supernatant was harvested after sufficient hemagglutination wasobserved. RNA was extracted from the harvested culture supernatant usinga QIAamp Viral RNA Mini Kit (QIAGEN catalog No. 52906), and subjected toRT-PCR that targets a region of the inserted gene. Whether the RT-PCRproduct has the correct nucleotide sequence was confirmed by sequencing.Thus, LmΔF/SeV Sendai virus vectors carrying various humantranscriptional factors were obtained.

The four reprogramming factors Oct3/4, Klf4, Sox2, and c-rMyc (TSΔF)were inserted into LmΔF/SeV at position 18+ in the same manner toconstruct viral vectors.

iPS cells were induced from human fibroblast BJ cells by infecting themwith LmΔF/SeV carrying the four factors in the same way as with the TSΔFSeV vectors. As a result, iPS-like colonies were formed in the samemanner as when using TSΔF/SeV, and the cells expressed ALP which is anES marker (FIG. 15A). This indicates that iPS can be induced using notonly one type of vector backbone, but also other Sendai virus vectorbackbones.

Example 14 Method for Inducing iPS without Feeder Cells

Because the Sendai virus vectors express the reprogramming factors athigh levels, iPS can be induced without feeder cells instead of usingthe conventional method of induction on feeder cells. Cells wereinfected with SeV carrying the reprogramming factors, and inducedwithout feeder in plastic dishes for 15 days after infection. WheniPS-like colonies were formed, the culture medium was changed fromDMEM/10% FBS to an ES cell medium. After the colonies becamesufficiently large, the cells were detached from the dishes usingcollagenase IV, and then plated onto fresh feeder cells. Thus, iPS cellscould be established.

Example 15 iPS Induction Using Thomson's Four Factors (Oct3/4, Sox2,Lin28, and Nanog)

iPS cells could also be induced from human fibroblasts by using TSΔF/SeVcarrying Thomson's four factors (Oct3/4, Sox2, Lin28, and Nanog) (Yu Jet al., Science. 2007, 318 (5858):1917-20), instead of Yamanaka's fourfactors (Oct3/4, Sox2, Klf4, and c-Myc) (Takahashi, K. and Yamanaka S.,Cell 126, 663-676, 2006) (FIG. 15B). An example of construction of Nanogand Lin28 vectors is described below.

(1) Isolation of the Human Transcriptional Factor Nanog, Construction ofa Sendai Virus Vector Plasmid Carrying Nanog, and Preparation of aSendai Virus Vector Carrying Nanog

A cDNA library of NCCIT cells was subjected to PCR using PrimeStar™ HSDNA polymerase (Takara Bio, catalog No. RO 10A) and the followingprimers: NANOF-F (5′-CCACCATGAGTGTGGATCCAGCTTGTCC-3′ (SEQ ID NO: 87))and NANOF-R (5′-CTCACACGTCTTCAGGTTGCATGTTC-3′ (SEQ ID NO: 88)). The PCRproduct was purified using a Qiaquick PCR Purification kit (QIAGEN,catalog No. 28106), and this was cloned into the EcoRV site of aBluescript plasmid vector. The gene sequence was determined bysequencing. A clone that has the correct sequence was selected, and thuspBS-KS-Nanog was obtained.

Then, PCR was carried out using pBS-KS-Nanog as a template, togetherwith the following primers: NotI-Nanog-F(5′-GCGCGGCCGCACCACCATGAGTGTGGATCCAGCTTGTCC-3′ (SEQ ID NO: 89)) andNotI-Nanog-R(5′-GCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCACACGTCTTCAGGTTGCATGTTCATGGAGTAGTTTAG-3′ (SEQ ID NO: 90)). The PCR product waspurified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and then this was digested with NotI. The digest was purifiedusing a Qiaquick PCR Purification kit (QIAGEN, catalog No. 28106), andthis was cloned into the NotI site of the pSeV18+/TSΔF vector. A clonethat has the correct sequence was selected by sequencing, and thuspSeV18+Nanog/TSΔF was obtained. Using this plasmid, an F gene-deficientSendai virus vector carrying the Nanog gene (hereinafter referred to as“SeV18+Nanog/TSΔF vector”) was prepared by the above-described method.

(2) Isolation of Human Lin28, Construction of a Sendai Virus VectorPlasmid Carrying Nanog, and Construction of a Sendai Virus VectorCarrying Lin28

A cDNA library of NCCIT cells was subjected to PCR using PrimeStar™ HSDNA polymerase (Takara Bio, catalog No. R010A) and the followingprimers: LIN28-F (5′-CCACCATGGGCTCCGTGTCCAACCAGC-3′ (SEQ ID NO: 91)) andLIN28-R (5′-GTCAATTCTGTGCCTCCGGGAGC-3′ (SEQ ID NO: 92)). The PCR productwas purified using a Qiaquick PCR Purification kit (QIAGEN, catalog No.28106), and this was cloned into the EcoRV site of a Bluescript plasmidvector. The gene sequence was determined by sequencing. A clone that hasthe correct sequence was selected, and thus pBS-KS-Lin28 was obtained.Then, PCR was carried out using pBS-KS-Lin28 as a template, togetherwith the following primers: NotI-Lin28-F(5′-GCGCGGCCGCACCACCATGGGCTCCGTGTCCAACCAGC-3′ (SEQ ID NO: 93)) andNotI-Lin28-R(5′-GCGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAATTCTGTGCCTCCGGGAGCAGGGTAGGGCTGTG-3′ (SEQ ID NO: 94)). The PCR product was purifiedusing a Qiaquick PCR Purification kit (QIAGEN, catalog No. 28106), andthen this was digested with NotI. The digest was purified using aQiaquick PCR Purification kit (QIAGEN, catalog No. 28106), and this wascloned into the NotI site of the pSeV18+/TSΔF vector. A clone that hasthe correct sequence was selected by sequencing, and thus pSeV18+Lin28/TSΔF was obtained. Using this plasmid, an F gene-deficient Sendaivirus vector carrying the Lin28 gene (herein referred to as “SeV18+Lin28/TSΔF vector”) was prepared by the above-described method.

INDUSTRIAL APPLICABILITY

The present invention allows production of ES-like cells (pluripotentstem cells) without integrating genes into the chromosome of host cells.Since no foreign gene is integrated into the chromosome of the producedcells, they are advantageous in tests and research. Furthermore, it isexpected that immunological rejection and ethical problems in diseasetreatments, as well as the risk of tumorigenesis due to genetic toxicitycan be avoided.

The invention claimed is:
 1. A Sendai virus vector having a deletion ofF gene, and encoding an M protein having mutations of G69E, T116A, andA183S, an HN protein having mutations of A262T, G264R, and K461G, a Pprotein having mutations of L511F, and a L protein having mutations ofN1197S and K1795E, wherein viral protein(s) encoded in said Sendai virusvector further comprises mutations of any one of (i) to (iv) below: (i)D433A, R434A and K437A in the P protein, and L1361C and L1558I in the Lprotein (ii) D433A, R434A and K437A in the P protein; (iii) Y942H,L1361C and L1558I in the L protein; and (iv) D433A, R434A and K437A inthe P protein, and L1558I in the L protein, and wherein the Sendai virusvector can be used for expressing reprograming factor genes in a somaticcell to induce an induced pluripotent stem cell.
 2. A method forintroducing one or more foreign genes into a single cell comprisingintroducing the Sendai virus vector according to claim 1 into the cell.3. The method according to claim 2, further comprising the step ofelevating the incubating temperature to remove said vectors.
 4. Themethod according to claim 3, wherein the incubating temperature iselevated to 37.5° C. to 39° C.
 5. A composition for introducing one ormore foreign genes into a single cell, comprising a carrier or diluentand the Sendai virus vector according to claim
 1. 6. The compositionaccording to claim 5, further comprising a Sendai virus vector having adeletion of F gene and encoding an M protein comprising the mutations ofG69E, T116A, and A183S, an HN protein comprising the mutations of A262T,G264R, and K461G, a P protein comprising the mutation of L511F, and an Lprotein comprising the mutations of N1197S and K1795E.